Vertebrate and nematode genes coding for yolk proteins are derived from a common ancestor.

One of the most obvious characteristics of the egg cells of oviparous animals is their large size resulting to a major extent from the deposition of nutritional reserves, mainly constituted of yolk proteins. In general, these are derived from a precursor called vitellogenin, which undergoes posttranslational modifications during secretion and during transport into and storage within the oocytes. Comparative analysis of the structural organization of the vitellogenin gene and of its product in different species shows that the vitellogenin gene is very ancient and that in vertebrates the gene may have more resemblance to the earliest gene than in invertebrates.

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription.

Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARgamma and PPARbeta, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARgamma and PPARbeta and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARbeta-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions.

The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse.

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

The embryonic blood-CSF barrier has molecular elements for specific glucose transport and for the general transport of molecules via transcellular routes.

In vertebrates, early brain development takes place at the expanded anterior end of the neural tube, which is filled with embryonic cerebrospinal fluid (E-CSF). We have recently identified a transient blood-CSF barrier that forms between embryonic days E3 and E4 in chick embryos and that is responsible for the transport of proteins and control of E-CSF homeostasis, including osmolarity. Here we examined the presence of glucose transporter GLUT-1 as well the presence of caveolae-structural protein Caveolin1 (CAV-1) in the embryonic blood-CSF barrier which may be involved in the transport of glucose and of proteins, water and ions respectively across the neuroectoderm. In this paper we demonstrate the presence of GLUT-1 and CAV-1 in endothelial cells of blood vessels as well as in adjacent neuroectodermal cells, located in the embryonic blood-CSF barrier. In blood vessels, these proteins were detected as early as E4 in chick embryos and E12.7 in rat embryos, i.e. the point at which the embryonic blood-CSF barrier acquires this function. In the neuroectoderm of the embryonic blood-CSF barrier, GLUT-1 was also detected at E4 and E12.7 respectively, and CAV-1 was detected shortly thereafter in both experimental models. These experiments contribute to delineating the extent to which the blood-CSF embryonic barrier controls E-CSF composition and homeostasis during early stages of brain development in avians and mammals. Our results suggest the regulation of glucose transport to the E-CSF by means of GLUT-1 and also suggest a mechanism by which proteins are transported via transcellular routes across the neuroectoderm, thus reinforcing the crucial role of E-CSF in brain development.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis.

Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling

Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca2+-dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca2+ content. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (<FONT FACE=Symbol>Dy</FONT>m). The collapse of <FONT FACE=Symbol>Dy</FONT>m along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway.

Methoxypyrazine removal in grape juice: Development of a removal system using odorant and pheromone binding proteins coupled with bentonite fining

Multicoloured Asian Lady Beetles (MALB) and 7-spot Lady Beetles that infect vineyards can secrete alkyl-methoxypyrazines when they are processed with the grapes, resulting in wines containing a taint. The main methoxypyrazine associated with this taint is 3-isopropyl-2-methoxypyrazine (IPMP). The wines are described as having aroma and flavours of peanut butter, peanut shells, asparagus and earthy which collectively, have become known as ���ladybug taint���. To date, there are no known fining agents used commercially added to juice or wine that are effective in removing this taint. The goal of this project was to use previously identified proteins with an ability to bind to methoxypyrazines at low pH, and subsequently develop a binding assay to test the ability of these proteins to bind to and remove methoxypyrazines from grape juice. The piglet odorant binding protein (plOBP) and mouse major urinary protein (mMUP) were identified, cloned and expressed in the Pichia pastoris expression system. Protein expression was induced using methanol and the proteins were subsequently purified from the induction media using anion exchange chromatography. The purified proteins were freeze-dried and rehydrated prior to use in the methoxypyrazine removal assay. The expression and purification system resulted in yields of approximately 78% of purified plOBP and 62% of purified mMUP from expression to rehydration. Purified protein values were 87 mg of purified plOPB per litre of induction media and 19 mg of purified mMUP per litre of induction medium. In order to test the ability of the protein to bind to the MPs, an MP removal assay was developed. In the assay, the purified protein is incubated with either IPMP or 3-isobutyl-2-methoxypyrazine (IBMP) for two hours in either buffer or grape juice. Bentonite is then used to capture the protein-MP complex and the bentonite-protein-MP complex is then removed from solution by filtration. Residual MP is measured in solution following the MP removal assay and compared to that in the starting solution by Gas Chromatography Mass Spectrometry (GC/MS). GC/MS results indicated that the mMUP was capable of removing IBMP and IPMP from 300 ng/L in buffer pH 4.0, buffer pH 3.5 and Riesling Juice pH 3.5 down to the limit of quantification of the instrument, which is 6ng/L and 2ng/L for IBMP and IPMP, respectively. The results for the plOBP showed that although it could remove some IBMP, it was only approximately 50-70 ng/L more than bentonite treatment followed by filtration, resulting in approximately 100 ng/L of the MPs being left in solution. pIOBP was not able to remove IPMP in buffer pH 3.5 using this system above that removed by bentonite alone. As well, the pIOBP was not able to remove any additional MPs from Chardonnay juice pH 3.5 above that already removed by the bentonite and filtration alone. The mouse MUP was shown to be a better candidate protein for removal of MPs from juice using this system.

Expression of steroidogenic proteins and genes in bovine placenta from conventional and somatic cell nuclear transfer (SCNT) gestations

Pendant la grossesse, les hormones st��ro��des jouent un r��le indispensable dans la r��gulation des principales manifestations physiologiques telles que la reconnaissance maternelle de la gestation, la r��ceptivit�� de l'endom��tre, le d��but du d��veloppement embryonnaire ainsi que le maintien de la gestation. Cependant, on sait tr��s peu sur la production de ces hormones et les principaux facteurs des voies intracellulaires impliqu��s dans le processus de st��ro��dogen��se dans le placenta bovin pendant les stades initiaux et plus avanc��s de la gestation. Par ailleurs, certaines anomalies du placenta chez les bovins suite �� une mauvaise production de st��ro��des n'ont pas encore ��t�� d��montr��es. Les objectifs de cette th��se ��taient donc de : 1) d��terminer la pr��sence et la localisation des principales prot��ines st��ro��diennes dans le placenta de bovins provenant de gestations de 50 �� 120 jours, 2) comparer l'expression placentaire d'une s��rie de g��nes et de prot��ines st��ro��diennes entre une gestation impliquant un transfert de noyaux de cellules somatiques (SCNT) et une gestation non-clonale; 3) ��tudier l'impact des hormones trophiques et des seconds messagers sur la st��ro��dogen��se dans le placenta bovin �� 140 +10 jours de gestation. L���utilisation de techniques d���immunohistochimie, d���immunobuvardage et de PCR quantitatif nous a permis d�����valuer la pr��sence d'un large ��ventail de g��nes st��ro��diens (STAR, CYP11A1, HSD3B1, CYP17A1 et SCARB1) qui participent au transport du cholest��rol et dans la production de diff��rents types de st��ro��des. Dans cette th��se, nous avons d��montr�� la capacit�� du placenta bovin d���initier la st��ro��dogen��se au d��but de la gestation et nous avons ��galement d��termin�� les principales cellules impliqu��es dans ce processus. Nous avons constat�� que les tissus maternels expriment les principaux marqueurs de st��ro��dogen��se sugg��rant une plus grande capacit�� st��ro��dog��nique que les tissus f��taux. En outre, un mod��le d'expression des prot��ines compl��mentaires st��ro��dog��niques entre la caroncule et le cotyl��don a ��t�� observ��, indiquant que la st��ro��dogen��se placentaire exige une communication cellule �� cellule entre les cellules de la m��re et du f��tus. Apr��s avoir d��montr�� les principales cellules impliqu��es dans la synth��se des hormones st��ro��diennes dans le placenta bovin en d��but de gestation, nous avons ensuite ��tudi�� les modifications possibles de la st��ro��dogen��se dans les tissus SCNT cotyl��donaires �� 40 jours de gestation. Nous avons identifi�� d'importantes modifications dans l'expression des g��nes STAR, CYP11A1, HSD3B1, CYP17A1, et SULT1E1. Cons��quemment, nous postulons que l'expression r��duite des g��nes st��ro��diens peut provoquer une insuffisance de la biosynth��se des hormones st��ro��diennes, ce qui pourrait contribuer �� un d��veloppement anormal du placenta et du f��tus dans les gestations SCNT �� court ou long terme. Finalement, nous avons d��velopp�� un mod��le efficace de culture d���explants de placentome qui nous a permis d'explorer les m��canismes sous-jacents sp��cifiques �� la st��ro��dogen��se placentaire. Nous avons explor�� l'effet stimulant des hormones trophiques et diff��rents messagers secondaires sur l'expression de diff��rentes prot��ines st��ro��dog��niques ainsi que le taux de progest��rone (P4) dans les explants de placentome. En utilisant les techniques de RIA et de PCR quantitatif, nous avons constat�� que m��me si les analogues de l'hormone lut��inisante (hCG) ont un effet stimulant sur plusieurs g��nes st��ro��diens, le calcium ionophore est le principal modulateur dans la synth��se de la P4. Ces r��sultats sugg��rent que dans le placenta bovin, la synth��se de la P4 est modul��e principalement par l'afflux de calcium intracellulaire, et apparemment les nucl��otides cycliques ne semblent pas contr��ler ce processus. En conclusion, cette ��tude contribue de mani��re significative �� une meilleure compr��hension des m��canismes d'entra��nement de la synth��se des st��ro��des placentaires au d��but de la gestation et permet aussi d���apporter de nouveaux ��clairages sur l'importance des st��ro��des placentaires dans la r��gulation du d��veloppement du placenta et du f��tus.

Axe et rotaxane parapluie : vers de nouveaux transporteurs transmembranaires de chlorures et de m��dicaments cycliques

La membrane cellulaire est principalement une bicouche phospholipidique constituant une barri��re qui r��gule les ��changes entre la cellule et son environnement. Son int��rieur hydrophobe emp��che le passage d���esp��ces hydrophiles, charg��es, de grande masse mol��culaire et polaires, qui sont g��n��ralement transport��es par des prot��ines �� travers la bicouche. Dans certains cas de syst��mes d��fectueux (e.g. les canalopathies), l�����quilibre des concentrations en ions �� l���int��rieur et �� l���ext��rieur des cellules est perturb�� et les cellules sont compromises. C���est pourquoi le d��veloppement de transporteurs transmembranaires synth��tiques est n��cessaire. De nombreux travaux ont ��t�� faits dans le d��veloppement de transporteurs synth��tiques d���anions (particuli��rement du chlorure). Dans cette th��se, nous pr��sentons nos travaux sur un nouveau transporteur d���anion appel�� axe parapluie, capable de changer de conformation d��pendamment de la polarit�� de son environnement. Dans un premier temps, nous avons con��u le design, puis synth��tis�� ces axes parapluie qui montrent une importante activit�� en tant que transporteur de chlorures. Ces compos��s r��unissent deux concepts : - Le parapluie, constitu�� d���acides biliaires amphiphiles (une face hydrophile, une face hydrophobe). La flexibilit�� des articulations combin��e �� la grande surface des acides choliques permettent d���emp��cher les interactions d��favorables entre les parties hydrophiles et hydrophobes, ce qui facilite l���insertion dans la bicouche. - Un site ammonium secondaire en tant que site de reconnaissance, capable de former des ponts hydrog��ne avec des ions chlorure. De plus, l���axe peut complexer une roue de type ��ther couronne pour former un pseudo-rotaxane ou rotaxane parapluie ce qui r��sulte en l���inhibition partielle de leurs propri��t��s de transport. Ceci nous m��ne au second objectif de cette th��se, le d��veloppement d���un nouveau moyen de transport pour les m��dicaments cycliques. Certains macrocycles polaires et biologiquement actifs tels que les nactines ont besoin d���atteindre leur objectif �� l���int��rieur de la cellule pour jouer leur r��le. La membrane cellulaire est alors un obstacle. Nous avons imagin�� tirer profit de notre axe parapluie pour transporter un m��dicament cyclique (en tant que roue d���un rotaxane parapluie). Les assemblages des rotaxanes parapluie furent accomplis par la m��thode de clipage. Le comportement de l���axe et du rotaxane parapluie fut ��tudi�� par RMN et fluorim��trie. Le mouvement du parapluie passant d���une conformation ferm��e �� expos��e d��pendamment du milieu fut observ�� pour le rotaxane parapluie. Il en fut de m��me pour les interactions entre le rotaxane parapluie et des v��sicules constitu��es de phospholipides. Finalement, la capacit�� du rotaxane parapluie �� franchir la bicouche lipidique pour transporter la roue �� l���int��rieur de la v��sicule fut d��montr��e �� l���aide de liposomes contenant de la ��-chymotrypsine. Cette derni��re pu cliver certains liens amide de l���axe parapluie afin de relarguer la roue.

R��gulation du m��tabolisme et du transport des lipides dans les macrophages : potentiel anti-ath��roscl��rotique des ligands du CD36

Les maladies cardiovasculaires sont la principale cause de morbidit�� et de mortalit�� dans les pays industrialis��s. Le r��cepteur CD36, exprim�� �� la surface des macrophages, joue un r��le d��terminant dans l���internalisation des lipoprot��ines oxyd��es menant �� la formation des cellules spumeuses dans l���espace sous endoth��lial, premi��re ��tape du d��veloppement des l��sions ath��roscl��rotiques. Nous avons montr�� pr��c��demment que les s��cr��tines de l���hormone de croissance sont des ligands du r��cepteur CD36 qui poss��dent un site de liaison qui chevauche celui des lipoprot��ines oxyd��es. Cependant, aucune ��tude n���avait rapport�� les effets potentiels des ligands s��lectifs du CD36 sur la progression des l��sions ath��roscl��rotiques et le m��tabolisme lipidique au niveau des macrophages. Ainsi, ce projet de doctorat visait �� ��valuer le potentiel anti-ath��roscl��rotique du EP 80317, un ligand s��lectif du CD36, et ��lucider les m��canismes �� l���origine de ses effets sur le m��tabolisme et le transport des lipides au niveau des macrophages. �� cette fin, des souris d��ficientes en apolipoprot��ine E (apoE-/-), nourries avec une di��te riche en lipides et en cholest��rol, ont ��t�� trait��es quotidiennement pendant 12 semaines avec le EP 80317, montrant un puissant effet anti-ath��roscl��rotique associ�� �� une r��duction de 51% des l��sions aortiques et de 30% du taux plasmatique de cholest��rol total. Cette m��me ��tude a permis de montrer une r��duction de l���internalisation des lipoprot��ines oxyd��es ainsi qu���une augmentation de l���expression des g��nes/prot��ines impliqu��s dans l���efflux du cholest��rol au niveau des macrophages, comme le peroxisome proliferator-activated receptor �� (PPAR��), liver x receptor �� (LXR��) et les transporteurs ABCA1 et ABCG1, entra��nant une r��duction de la formation des cellules spumeuses. Ces observations nous ont conduits �� ��lucider les m��canismes mol��culaires engendr��s par la liaison d���un ligand s��lectif au r��cepteur CD36 dans les macrophages. Les ��tudes ont permis de montrer que les ligands du CD36 entra��nent une augmentation de l���efflux du cholest��rol vers les transporteurs ABCA1 et ABCG1 en augmentant l���expression prot��ique de la cyclooxyg��nase 2 (COX-2) cons��cutive �� la phosphorylation de la MAP kinase ERK1/2. L���activation de COX-2 stimule la production intracellulaire de la prostaglandine 15d-PGJ2, cette derni��re conduisant �� l���activation du PPAR��. Finalement, une troisi��me ��tude nous a permis de mettre en ��vidence les effets du EP 80317 sur le transport inverse du cholest��rol in vivo. L���injection de macrophages J774 radiomarqu��s avec du cholest��rol triti�� dans la cavit�� p��riton��ale de souris avec le EP 80317 nous a permis de montrer que le EP 80317 entra��ne une r��duction de la radioactivit�� retrouv��e dans le foie tandis qu���il augmente celle retrouv��e dans les f��ces par comparaison aux souris contr��les, sans n��anmoins modifier le profil plasmatique du radiotraceur entre les deux groupes. De plus, l���expression des g��nes impliqu��s dans le transport du cholest��rol au niveau intestinal comme le LXR��, ABCA1, ABCG5 ainsi que ABCG8 ont ��t�� r��gul��s �� la hausse par le EP 80317 tandis que l���expression de NPC1L1, un transporteur impliqu�� dans l���absorption du cholest��rol, a ��t�� r��gul�� �� la baisse. Toutefois, les g��nes impliqu��s dans le m��tabolisme du cholest��rol au niveau du foie ne sont pas modul��s par le EP 80317. En conclusion, les travaux effectu��s dans le cadre de cette th��se nous ont permis de montrer que l���activation du r��cepteur CD36 par le EP 80317 pourrait s���av��rer ��tre une nouvelle approche th��rapeutique pour le traitement de l���ath��roscl��rose. Les effets anti-ath��roscl��rotiques et hypocholest��rol��miants des ligands synth��tiques du r��cepteur CD36 sont en partie engendr��s par 1) la r��gulation du m��tabolisme des lipides au niveau des macrophages en r��ponse �� l���activation du PPAR�� par son ligand endog��ne, le 15d-PGJ2 et 2) par une augmentation du transport inverse du cholest��rol, particuli��rement par une augmentation de l���efflux transintestinal.

Biochemical effects of cadmium, salinity and temperature on the catabolism of proteins and purines in oreochromis mossambicus (Peters)

In the present investigation, three important stressors: cadmium ion (Cd++), salinity and temperature were selected to study their effects on protein and purine catabolism of O. mossambicus. Cadmium (Cd) is a biologically nonessential metal that can be toxic to aquatic animals. Cadmium is a trace element which is a common constituent of industrial effluents. It is a non-nutrient metal and toxic to fish even at low concentrations. Cadmium ions accumulate in sensitive organs like gills, liver, and kidney of fish in an unregulated manner . Thus; the toxic effects of cadmium are related to changes in natural physiological and biochemical processes in organism. The mechanics of osmoregulation (i.e. total solute and water regulation) are reasonably well understood (Evans, 1984, 1993), and most researchers agree that salinities that differ from the internal osmotic concentration of the fish must impose energetic regulatory costs for active ion transport. There is limited information on protein and purine catabolism of euryhaline fish during salinity adaptation. Within a range of non-lethal temperatures, fishes are generally able to cope with gradual temperature changes that are common in natural systems. However, rapid increases or decreases in ambient temperature may result in sub lethal physiological and behavioral responses. The catabolic pathways of proteins and purines are important biochemical processes. The results obtained signifies that O. mossambicus when exposed to different levels of cadmium ion, salinity and temperature show great variation in the catabolism of proteins and purines. The organism is trying to attain homeostasis in the presence of stressors by increasing or decreasing the activity of certain enzymes. The present study revealed that the protein and purine catabolism in O. mossambicus is sensitive to environmental stressors.

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component���the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ���HxxE��� motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ���EfeO-Cup��� family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.