Facilitated glucose transporters in epithelial cells.

The molecular cloning of facilitated sugar transporters has led to the identification of a family of transport molecules having similar functions, but possessing specific kinetic and regulatory properties. These transporter isoforms are characterized by different primary structures, specific tissue localization, and polarized expression within the same epithelial cells. The use of Xenopus oocytes for the functional expression of different members of this transporter family has been of considerable value in defining the kinetic properties and sugar specificities of the different isoforms. The expression of chimeric or variously mutated transporters should, in the near future, permit the determination of the structural basis for their kinetic properties and sugar specificities. cDNA probes and antipeptide antibodies specific for each isoform are now being used to determine their specific regulation during development and in different states of altered glucose homeostasis. The variety of molecular forms implicated in the apparently simple task of sugar uptake or transepithelial transport has been surprising. With the available molecular tools now in hand, it will be possible to study these mechanisms in much greater detail.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Law and the practise of principle in diverse societies

Projecte de recerca elaborat a partir d’una estada a la London School of Economics and Political Science, United Kingdom, entre 2007 i 2009. L’objecte principal del projecte ha estat analitzar les implicacions jurídico-polítiques i institucionals d’una teoria de la justícia i la igualtat liberals aplicada a societats multiculturals amb un marcat predomini de la diversitat cultural. L’anàlisi desenvolupa una línia d'investigació interdisciplinar - entre el dret i la teoria política - iniciada en una tesis doctoral sobre multiculturalisme i drets de les minories culturals (UPF, 2000) que va culminar en la publicació de Group Rights as Human Rights (Springer, 2006). La recerca adopta com a punt de partida les conclusions de l'esmentada obra, en especial, la rellevància del reconeixement de drets col•lectius; tanmateix, el tipus de qüestions plantejades, l’enfoc i la metodologia emprades són substancialment diferents. En concret, s'adrecen preguntes específiques sobre el model i aspiracions del constitucionalisme democràtic i el paper del dret en contextos multiculturals. També s’atorga un pes central a la dimensió institucional dels models de gestió de la diversitat que s’analitzen, prioritzant un enfocament comparatiu a partir de l’estudi de controvèrsies concretes. L’objectiu és superar algunes limitacions importants de la literatura actual, com ara la tendència a examinar en abstracte la compatibilitat de determinades demandes amb el constitucionalisme democràtic, sense abordar el funcionament d'estratègies de gestió de la diversitat cultural emprades en contextos concrets. Els treballs producte d'aquest projecte articulen les línies bàsiques d’un model pluralista, basat en principis més que en regles, que desafia els plantejaments dominants actualment. Aquest model es caracteritza pel compromís amb la legitimitat i igualtat comparatives, rebutjant el paternalisme i les visions liberals típiques sobre el paper de la regulació. La presumpció de l’“standing” moral dels grups identitaris és fonamental per tal de considerar-los interlocutors vàlids amb interessos genuïns. També s’argumenta que la integració social en contextos multiculturals no depèn tant de l’eliminació del conflicte sinó, sobre tot, d’una gestió eficient que eviti abusos de poder sistemàtics. El model defensa el rol del dret en la institucionalització del diàleg intercultural, però admet que el diàleg no necessàriament condueix a l’acord o a una estructura reguladora coherent i uniforme. Les aspiracions del ordre jurídic pluralista són més modestes: afavorir la negociació i resolució en cada conflicte, malgrat la persistència de la fragmentació i la provisionalitat dels acords. La manca d'un marc regulador comú esdevé una virtut en la mesura que permet la interacció de diferents subordres; una interacció governada per una multiplicitat de regles no necessàriament harmòniques. Els avantatges i problemes d’aquest model s'analitzen a partir de l'anàlisi de l’estructura fragmentària de l'ordre jurídic internacional i del règim Europeu de drets humans.

FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes.

Recently, corticosteroid hormone-induced factor (CHIF) and the gamma-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase alpha 1-beta 1, alpha 2-beta 1 and alpha 3-beta 1 but not with alpha-beta 2 isozymes, whereas, in brain, it is only associated with alpha 1-beta isozymes. FXYD7 decreases the apparent K(+) affinity of alpha 1-beta 1 and alpha 2-beta 1, but not of alpha 3-beta1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.

Does VAA popularity pay off ? : Smartvote users preferred candidates and their results in the 2007 Swiss federal elections

Voting is fundamental for democracy, however, this decisive democratic act requires quite an effort. Decision making at elections depends largely on the interest to gather information about candidates and parties, the effort to process the information at hand and the motivation to reach a vote choice. Especially in electoral systems with highly fragmented party systems and hundreds of candidates running for office, the process of decision making in the pre‐election sphere is highly demanding. In the age of information and communication technologies, new possibilities for gathering and processing such information are available. Voting Advice Applications (VAAs) provide guidance to voters prior to the act of voting and assist voters in choosing between different candidates and parties on the basis of issue congruence. Meanwhile widely used all over the world, scientific inquiry into the effect of such tools on electoral behavior is ongoing. This paper adds to the current debate by focusing on whether the popularity of candidates on the Swiss VAA smartvote eventually paid off at the 2007 Swiss federal elections and whether there is a direct link between the performance of a candidate on the tool and his or her electoral performance.

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.

Laboratory estimation of the effects of increasing temperatures on the duration of gonotrophic cycle of Anopheles albimanus (Diptera: Culicidae)

The increase of malaria transmission in the Pacific Coast of Colombia during the occurrence of El Niño warm event has been found not to be linked to increases in the density of the vector Anopheles albimanus, but to other temperature-sensitive variables such as longevity, duration of the gonotrophic cycle or the sporogonic period of Plasmodium. The present study estimated the effects of temperature on duration of the gonotrophic cycle and on maturation of the ovaries of An. albimanus. Blood fed adult mosquitoes were exposed to temperatures of 24, 27, and 30°C, held individually in oviposition cages and assessed at 12 h intervals. At 24, 27, and 30°C the mean development time of the oocytes was 91.2 h (95% C.I.: 86.5-96), 66.2 h (61.5-70.8), and 73.1 h (64-82.3), respectively. The mean duration of the gonotrophic cycle for these three temperatures was 88.4 h (81.88-94.9), 75 h (71.4-78.7), and 69.1 h (64.6-73.6) respectively. These findings indicate that both parameters in An. albimanus are reduced when temperatures rose from 24 to 30°C, in a nonlinear manner. According to these results the increase in malaria transmission during El Niño in Colombia could be associated with a shortening of the gonotrophic cycle in malaria vectors, which could enhance the frequency of man-vector contact, affecting the incidence of the disease.

What is the most relevant standard of success in assisted reproduction?: The cumulated singleton/twin delivery rates per oocyte pick-up: the CUSIDERA and CUTWIDERA.

National and international registries are essential tools for establishing new standards and comparing success rates, but they do not take into account the total pregnancy/delivery rate per oocyte recovery. In Switzerland and Germany, because of legal constraints, a maximum of three two-pronuclear zygotes are allocated for transfer whereas all the supernumerary pronuclear zygotes are immediately cryopreserved, preventing selection of the transferred embryos. We report on a 10 years' experience (1993-2002) of our centre which performs transfers of unselected embryos and cryopreservation at the two-pronuclear zygote stage. As approximately 30% of all deliveries are from cryo cycles, it is essential to take into account the contribution of the cryo transfers, and we propose therefore to evaluate, as a measure of IVF performance, the cumulated delivery rate per oocyte pick-up. This delivery rate is broken down further into the cumulated singleton delivery rate (CUSIDERA) and the cumulated twin delivery rate (CUTWIDERA). The sum (S) of these two rates is a measure of efficacy while the ratio CUTWIDERA/S as a percentage is a measure of safety of IVF treatments. Using these new indexes, the average 10 year efficacy and safety of our IVF programme were 26 and 19%, respectively. Both CUSIDERA and CUTWIDERA can be calculated easily in any clinical situation and yield useful parameters for patient counselling and internal/external benchmarking purposes.

The transmembrane serine protease (TMPRSS3) mutated in deafness DFNB8/10 activates the epithelial sodium channel (ENaC) in vitro.

TMPRSS3 encodes a transmembrane serine protease that contains both LDLRA and SRCR domains and is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). To study its function, we cloned the mouse ortholog which maps to Mmu17, which is structurally similar to the human gene and encodes a polypeptide with 88% identity to the human protein. RT-PCR and RNA in situ hybridization on rat and mouse cochlea revealed that Tmprss3 is expressed in the spiral ganglion, the cells supporting the organ of Corti and the stria vascularis. RT-PCR on mouse tissues showed expression in the thymus, stomach, testis and E19 embryos. Transient expression of wild-type or tagged TMPRSS3 protein showed a primary localization in the endoplasmic reticulum. The epithelial amiloride-sensitive sodium channel (ENaC), which is expressed in many sodium-reabsorbing tissues including the inner ear and is regulated by membrane-bound channel activating serine proteases (CAPs), is a potential substrate of TMPRSS3. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. In contrast, 6 TMPRSS3 mutants (D103G, R109W, C194F, W251C, P404L, C407R) causing deafness and a mutant in the catalytic triad of TMPRSS3 (S401A), failed to undergo proteolytic cleavage and activate ENaC. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage and suggest: (i) the existence of an auto-catalytic processing by which TMPRSS3 would become active, and (ii) that ENaC could be a substrate of TMPRSS3 in the inner ear.

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit.

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.

The future of television : advertising, technology and the pursuit of audiences

La televisió està en crisi. Les noves tecnologies i els dispositius han fragmentat les audiències de televisió en segments més petits. En aquest informe, Marissa Gluck i Meritxell Roca Sales examinen l'explosió dels mitjans de comunicació que han impulsat aquesta fragmentació.

Randomized controlled trial comparing highly purified (HP-hCG) and recombinant hCG (r-hCG) for triggering ovulation in ART.

BACKGROUND: A randomized controlled trial (RCT) comparing highly purified human Choriogonadotrophin (HP-hCG) and recombinant hCG (r-hCG) both administered subcutaneously for triggering ovulation in controlled ovarian stimulation (COS) for Assisted Reproductive Technology (ART). METHODS: Multi-centre (n = 4), prospective, controlled, randomized, non-inferiority, parallel group, investigator blind design, including 147 patients. The trial was registered with www.clinicaltrials.gov, using the identifier: NCT00335569. The primary endpoint is the number of oocytes retrieved, while the secondary endpoints include embryo implantation, pregnancy and delivery rates as well as safety parameters. RESULTS: The number of retrieved oocytes was not inferior when HP-hCG was used as compared to r-hCG: the mean number was 13.3 (6.8) in HP-hCG and 12.5 (5.8) in the r-hCG group (p = 0.49) with a 95% CI (-1.34, 2.77). Regarding the secondary outcomes, there were also no differences in fertilization rate at 57.3% (467/815) vs. 61.3% (482/787) (p = 0.11), the number of embryos available for transfer and cryopreservation (2PN stage) and implantation, pregnancy and delivery rates. Furthermore, there were no differences in the number and type of adverse events reported. HP-hCG was therefore not inferior to r-hCG. CONCLUSIONS: HP-hCG and r-hCG are equally efficient and safe for triggering ovulation in ART and, both being administered subcutaneously, equally practical and well tolerated by patients.

Genotypic features of lentivirus transgenic mice.

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

La transformació del paisatge del delta de la Tordera en els darrers cent cinquanta anys : una anàlisi per mitjà dels canvis en els usos i les cobertes del sòl

This article compares the mid-nineteenth century landscape of the River Tordera delta with the present day landscape, based, above all, on the changes that have occurred in land use and land cover. The mid 19th century landscape was reconstructed using data obtained from the amillaraments (land inventories) and other historical documents. Present-day land use and cover was established through photo interpretation and field work. The most important changes detected concern the almost complete disappearance of certain crops, such as vineyards, which were very important in the 19th century; the expansion of forest in place of abandoned tilled land and the increase in built up areas, which, taken together, produce a highly fragmented landscape pattern

Back to the future in Chagas disease: from animal models to patient cohort studies, progress in immunopathogenesis research

Despite the wealth of information generated by trans-disciplinary research in Chagas disease, knowledge about its multifaceted pathogenesis is still fragmented. Here we review the body of experimental studies in animal models supporting the concept that persistent infection by Trypanosoma cruzi is crucial for the development of chronic myocarditis. Complementing this review, we will make an effort to reconcile seemingly contradictory results concerning the immune profiles of chronic patients from Argentina and Brazil. Finally, we will review the results of molecular studies suggesting that parasite-induced inflammation and tissue damage is, at least in part, mediated by the activities of trans-sialidase, mucin-linked lipid anchors (TLR2 ligand) and cruzipain (a kinin-releasing cysteine protease). One hundred years after the discovery of Chagas disease, it is reassuring that basic and clinical research tends to converge, raising new perspectives for the treatment of chronic Chagas disease.