Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

Competition between beta-subunits of cardiac L-type calcium channels

Cardiac L-type Ca (CaV1.2) channels are composed of a pore forming CaV1.2-α1 subunit and auxiliary β- and α2δ-subunits. β-subunits are important not only for surface expression of the channel pore but also for modulation of channel gating properties. Different β-subunits differentially modulate channel activity (Hullin et al., PLOSone, 2007) and thus L-type Ca2+ channel gating is altered when β-subunit expression pattern is changed. In human heart failure increased activity of single ventricular L-type Ca2+-channels is associated with an increased expression of β2-subunits. Interestingly, induction of β2-subunit over-expression in hearts of transgenic mice resembled this heart failure phenotype of hyperactive single L-type Ca2+-channel channels (Beetz et al., Cardiovasc Res. 2009). We hypothesised that competition of less stimulating β-subunits (e.g. β1) with β-subunits causing strong channel stimulation (e.g. β2) might be a means to treat dysfunctional L-type Ca2+-channel activity. To test this hypothesis, we performed whole-cell and single-channel measurements employing recombinant CaV1.2 channels expressed in HEK293 cells together with both β- and β1a2b-subunits. Whole-cell analysis revealed no differences of maximum L-type Ca2+-current densities [pA/pF] with coexpression of either β1a-subunits (-52±3.8), β2b-subunits (-61.5±6.6) or the mixtures of β- and β1a2b-subunits with the plasmid transfection ratio of 2:1 (-60.2±1.6) and 1:1 (-56.7±2.6) respectively. However, steady state inactivation kinetics differed between particular β-subunit and the relative amount of β-subunit presence in the mixtures (β1a1a-subunit (-41.1±1.0), β2b-subunits (-35.1±1.1), mixture 2:1 (-40.3±1.5), and mixture 1:1 (-38.4±2.0); [mV]; p<0.05, students t-test). Using a novel single-channel analysis, switching of gating between β1-like and β2-like modes was monitored on a minute time-scale when both β-subunits were co-expressed in the same cells, but the larger amount of β1a-subunits is required for the effective switching of gating. Our results indicate a model of mutually exclusive binding and effective competition between several β-subunits suggesting that hyperactive channel gating mediated e.g. by β2-subunits can be normalized by β1-subunits. Therefore, competitive replacement between different L-type Ca2+-channel β-subunits might serve as a novel therapeutic strategy for e.g. heart failure.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions.

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8+ T-lymphocyte subsets, RA+CCR7-27+28+ and RA+CCR7-27+28-, in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon gamma, and tumor necrosis factor alpha. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

Rôle des facteurs physico-chimiques du micro-environnement intestinal et des boucles inter-hélicales du Domaine I dans l’activité de la toxine insecticide Cry9Ca du bacille de Thuringe

Une fois ingérées par un insecte sensible, les toxines insecticides du bacille de Thuringe doivent être activées par les protéases intestinales de cet insecte. Leur premier domaine, un ensemble de sept hélices-α amphipathiques, est responsable de leur insertion dans la membrane luminale de certaines cellules de l’intestin médian, ce qui crée des pores peu sélectifs. La toxicité et la capacité à former des pores d’une telle toxine, la Cry9Ca, de ses mutants simples R164A et R164K et d’un fragment de 55 kDa résultant d’un clivage protéolytique au niveau de son résidu 164 ont été étudiées à l’aide d’une combinaison de modélisation par homologie, de bioessais, d’expériences de gonflement osmotique avec des vésicules de membrane en bordure en brosse de larves de sphinx du tabac et de mesures électrophysiologiques sur des intestins isolés. Ni les mutations simples ni le clivage protéolytique n’ont altéré la toxicité de la Cry9Ca. Dans une solution à faible force ionique, toutefois, la formation des pores dépend fortement du pH : une augmentation de celui-ci de 6,5 à 10,5 a entraîné une baisse irrégulière et par étapes successives de la perméabilité membranaire. Les quatre préparations de toxine ont néanmoins dépolarisé la membrane apicale d’intestins médians fraîchement isolés baignant dans une solution contenant 122 mM de KCl à pH 10,5. L’activité de la Cry9Ca, et des mutants R164A et R164K, a été grandement stimulée lorsque les expériences ont été effectuées en présence de suc intestinal, de lipides extraits d’un volume équivalent de suc intestinal ou d’un cocktail d’inhibiteurs de protéases solubles dans l’eau. De plus, le rôle des boucles inter-hélicales du Domaine I lors de l’insertion dans la membrane a été étudié avec des mutants doubles de la Cry9Ca dont les mutations introduisaient, neutralisaient ou renversaient une charge électrique. À l’exception de trois d’entres eux, tous ces mutants ont conservé une toxicité et une capacité à former des pores comparables à celles de la toxine parentale. L’ensemble de ces résultats suggère que le micro-environnement de l’intestin médian contribue à minimiser l’influence des charges de surface portées par les résidus des boucles inter-hélicales du Domaine I sur la capacité des toxines du bacille de Thuringe à former des pores. Il indique aussi que, d’une part, selon le site de clivage et les conditions expérimentales utilisées, des protéolyses supplémentaires de la toxine Cry9Ca activée peuvent soit stimuler, soit nuire à son activité et que, d’autre part, le suc intestinal du sphinx du tabac contient probablement un inhibiteur de protéases qui pourrait jouer un rôle important dans l’activité des toxines du bacille de Thuringe.

Genome wide search for genetic determinants of habitual alcohol, tobacco and coffee use, obesity-related traits, response to mental and physical stress and hemodynamic traits

Les habitudes de consommation de substances psychoactives, le stress, l’obésité et les traits cardiovasculaires associés seraient en partie reliés aux mêmes facteurs génétiques. Afin d’explorer cette hypothèse, nous avons effectué, chez 119 familles multi-générationnelles québécoises de la région du Saguenay-Lac-St-Jean, des études d’association et de liaison pangénomiques pour les composantes génétiques : de la consommation usuelle d’alcool, de tabac et de café, de la réponse au stress physique et psychologique, des traits anthropométriques reliés à l’obésité, ainsi que des mesures du rythme cardiaque (RC) et de la pression artérielle (PA). 58000 SNPs et 437 marqueurs microsatellites ont été utilisés et l’annotation fonctionnelle des gènes candidats identifiés a ensuite été réalisée. Nous avons détecté des corrélations phénotypiques significatives entre les substances psychoactives, le stress, l’obésité et les traits hémodynamiques. Par exemple, les consommateurs d’alcool et de tabac ont montré un RC significativement diminué en réponse au stress psychologique. De plus, les consommateurs de tabac avaient des PA plus basses que les non-consommateurs. Aussi, les hypertendus présentaient des RC et PA systoliques accrus en réponse au stress psychologique et un indice de masse corporelle (IMC) élevé, comparativement aux normotendus. D’autre part, l’utilisation de tabac augmenterait les taux corporels d’épinéphrine, et des niveaux élevés d’épinéphrine ont été associés à des IMC diminués. Ainsi, en accord avec les corrélations inter-phénotypiques, nous avons identifié plusieurs gènes associés/liés à la consommation de substances psychoactives, à la réponse au stress physique et psychologique, aux traits reliés à l’obésité et aux traits hémodynamiques incluant CAMK4, CNTN4, DLG2, DAG1, FHIT, GRID2, ITPR2, NOVA1, NRG3 et PRKCE. Ces gènes codent pour des protéines constituant un réseau d’interactions, impliquées dans la plasticité synaptique, et hautement exprimées dans le cerveau et ses tissus associés. De plus, l’analyse des sentiers de signalisation pour les gènes identifiés (P = 0,03) a révélé une induction de mécanismes de Potentialisation à Long Terme. Les variations des traits étudiés seraient en grande partie liées au sexe et au statut d’hypertension. Pour la consommation de tabac, nous avons noté que le degré et le sens des corrélations avec l’obésité, les traits hémodynamiques et le stress sont spécifiques au sexe et à la pression artérielle. Par exemple, si des variations ont été détectées entre les hommes fumeurs et non-fumeurs (anciens et jamais), aucune différence n’a été observée chez les femmes. Nous avons aussi identifié de nombreux traits reliés à l’obésité dont la corrélation avec la consommation de tabac apparaît essentiellement plus liée à des facteurs génétiques qu’au fait de fumer en lui-même. Pour le sexe et l’hypertension, des différences dans l’héritabilité de nombreux traits ont également été observées. En effet, des analyses génétiques sur des sous-groupes spécifiques ont révélé des gènes additionnels partageant des fonctions synaptiques : CAMK4, CNTN5, DNM3, KCNAB1 (spécifique à l’hypertension), CNTN4, DNM3, FHIT, ITPR1 and NRXN3 (spécifique au sexe). Ces gènes codent pour des protéines interagissant avec les protéines de gènes détectés dans l’analyse générale. De plus, pour les gènes des sous-groupes, les résultats des analyses des sentiers de signalisation et des profils d’expression des gènes ont montré des caractéristiques similaires à celles de l’analyse générale. La convergence substantielle entre les déterminants génétiques des substances psychoactives, du stress, de l’obésité et des traits hémodynamiques soutiennent la notion selon laquelle les variations génétiques des voies de plasticité synaptique constitueraient une interface commune avec les différences génétiques liées au sexe et à l’hypertension. Nous pensons, également, que la plasticité synaptique interviendrait dans de nombreux phénotypes complexes influencés par le mode de vie. En définitive, ces résultats indiquent que des approches basées sur des sous-groupes et des réseaux amélioreraient la compréhension de la nature polygénique des phénotypes complexes, et des processus moléculaires communs qui les définissent.

Étude des mécanismes moléculaires de formation des pores des toxines formeuses de pores par la spectroscopie de fluorescence

Les toxines formeuses de pore (PFTs) sont des protéines exogènes responsables d’un grand nombre de maladies infectieuses qui perméabilisent les membranes cellulaires de leur hôte. La formation des pores ou l’introduction d’une enzyme dans le cytoplasme peut entrainer l’apparition de symptômes de maladies connues (l’anthrax, le botulisme) et, dans le pire des cas, la mort. Les mécanismes d’infection et de destruction des cellules infectées sont bien caractérisés. Toutefois, l’aspect dynamique des changements de conformation durant le processus de perméabilisation reste à découvrir pour la majorité des toxines formeuses de pore. Le but de cette thèse est d’étudier les mécanismes d’oligomérisation des PFTs, ainsi que la formation des pores à la membrane lipidique grâce à la spectroscopie de fluorescence. Nous avons choisi la toxine Cry1Aa, un bio pesticide produit par le bacille de Thuringe et qui a été rigoureusement caractérisé, en tant que modèle d’étude. La topologie de la Cry1Aa à l’état actif et inactif a pu être résolue grâce à l’utilisation d’une technique de spectroscopie de fluorescence, le FRET ou transfert d’énergie par résonance entre un fluorophore greffé au domaine formeur de pore (D1) et un accepteur non fluorescent (le DPA ou dipicrylamine) localisé dans la membrane et qui bouge selon le potentiel membranaire. Le courant électrique, ainsi que la fluorescence provenant de la bicouche lipidique membranaire horizontale ont été enregistrés simultanément. De cette manière, nous avons pu localiser toutes les boucles reliant les hélices de D1 avant et après la formation des pores. Dans la forme inactive de la toxine, toutes ces boucles se trouvent du côté interne de la bicouche lipidique, mais dans sa forme active l’épingle α3-α4 traverse du côté externe, alors que toutes les autres hélices demeurent du côté interne. Ces résultats suggèrent que α3-α4 forment le pore. Nous avons découvert que la toxine change significativement de conformation une fois qu’elle se trouve dans la bicouche lipidique, et que la Cry1Aa attaque la membrane lipidique de l’extérieur, mais en formant le pore de l’intérieur. Dans le but de caractériser la distribution de toxines à chaque extrémité de la bicouche, nous avons utilisé une technique de double FRET avec deux accepteurs ayant des vitesses de translocation différentes (le DPA et l’oxonol) dans la membrane lipidique. De cette manière, nous avons déterminé que la toxine était présente des deux côtés de la bicouche lipidique durant le processus de perméabilisation. La dynamique d’oligomérisation de la toxine dans une bicouche lipidique sans récepteurs a été étudiée avec une technique permettant le compte des sauts de fluorescence après le photoblanchiment des fluorophore liés aux sous unités composant un oligomère présent dans la bicouche lipidique supportée. Nous avons confirmé de cette manière que la protéine formait ultimement des tétramères, et que cet état résultait de la diffusion des monomères de toxine dans la bicouche et de leur assemblage subséquent. Enfin nous avons voulu étudier le « gating » de la colicine Ia, provenant de la bactérie E.Coli, dans le but d’observer les mouvements que font deux positions supposées traverser la bicouche lipidique selon le voltage imposé aux bornes de la bicouche. Nos résultats préliminaires nous permettent d’observer un mouvement partiel (et non total) de ces positions, tel que le suggèrent les études de conductances du canal.

Nanoshells and nanotubes from block copolymers

Recent work exploring the use of block copolymer vesicles and tubules is reviewed. The stability and toughness of block copolymer vesicles are enhanced compared to those formed by low molar mass amphiphiles. Functionality can also readily be introduced through the polymer chemistry or by incorporating additional components (for example pore-forming membrane proteins). This design flexibility leads to numerous potential applications in encapsulation, in targeted drug delivery, templating of inorganic materials and many others.

Ion channels as effectors in carbon monoxide signaling

A wealth of recent studies has highlighted the diverse and important influences of carbon monoxide (CO) on cellular signaling pathways. Such studies have implicated CO, and the enzymes from which it is derived (heme oxygenases) as potential therapeutic targets, particularly (although not exclusively) in inflammation, immunity and cardiovascular disease.1 In a recent study,2 we demonstrated that CO inhibited cardiac L-type Ca(2+) channels. This effect arose due to the ability of CO to bind to mitochondria (presumably at complex IV of the electron transport chain) and so cause electron leak, which resulted in increased production of reactive oxygen species. These modulated the channel's activity through interactions with three cysteine residues in the cytosolic C-terminus of the channel's major, pore-forming subunit. Our study provided a potential mechanism for the cardioprotective effects of CO and also highlighted ion channels as a major potential target group for this gasotransmitter.

Relationship Between Expression of Voltage-Dependent Anion Channel (VDAC) Isoforms and Type of Hexokinase Binding Sites on Brain Mitochondria

Voltage-dependent anion channels (VDAC) are pore-forming proteins found in the outer mitochondrial membrane of eukaryotes. VDACs are known to play an essential role in cellular metabolism and in early stages of apoptosis. In mammals, three VDAC isoforms have been identified. A proteomic approach was exploited to study the expression of VDAC isoforms in rat, bovine, and chicken brain mitochondria. Given the importance of mitochondrially bound hexokinase in regulation of aerobic glycolysis in brain, we studied the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A/type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was significantly higher in bovine than in rat brain. Chicken brain mitochondria showed the highest VDAC1 expression and the lowest of VDAC2. Bovine brain mitochondria had the highest VDAC2 levels. We concluded that the nature of hexokinase binding site is not determined by the expression of a single VDAC isoform.

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats A Novel Insight Into Vascular Dysfunction

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 mu mol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 mu mol/L) or gadolinium (100 mu mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension. (Hypertension. 2009; 53[part 2]: 409-416.)

EFFECT of DIFFERENT SOLID AMOUNTS IN THE PHYSICAL and MICROSCOPICAL PROPERTIES of SiC POROUS CERAMICS

The presence of pores in ceramics is directly related to the chosen forming process. So, in the starch consolidation method, the ceramics show, after burning, pores with morphology similar to that presented by this organic material. on the other hand, the increase in solid load leads up to alterations in dispersion viscosity, increasing the thermal stresses during drying and sintering processes. In order to verify the solid percentage influence in ceramic final properties, samples were prepared with silicon carbide in different compositions using or not starch as binder agent and pore forming element. The characterization of the ceramic pieces was performed by superficial roughness measurements, porosity besides by optical and scanning electron microscopy. The results showed ceramics with SiC and starch presented physical and microscopic properties slightly higher in relation to those with only ceramic powder in their composition. The presence of organic material, agglomerated and foam during the forming were essential for the final properties of the studied samples.

Alumina porous ceramics: mathematical behavior at different commercial starch concentration

Several researches have been developed in order to verify the porosity effect over the ceramic material properties. The starch consolidation casting (SCC) allows to obtain porous ceramics by using starch as a binder and pore forming element. This work is intended to describe the porous mathematical behavior and the mechanical resistance at different commercial starch concentration. Ceramic samples were made with alumina and potato and corn starches. The slips were prepared with 10 to 50 wt% of starch. The specimens were characterized by apparent density measurements and three-point flexural test associated to Weibull statistics. Results indicated that the porosity showed a first-order exponential equation e(-x/c) increasing in both kinds of starches, so it was confirmed that the alumina ceramic porosity is related to the kind of starch used. The mechanical resistance is represented by a logarithmic expression R = A + B/1+10((Log(x0)-P)C).