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The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine- rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter. [ABSTRACT FROM AUTHOR]

Past position and subsequent tectonic evolution of the Chortis Block represents one of the major uncertainties related to the geodynamic evolution of southern Mexico and northern Central America. This uncertainty has led to different hypotheses and evolutionary models based on tectonic, geological and paleographic studies that attempt to explain the origin and paleoposition of the Chortis Block. Among these models, a challenging hypothesis suggests the Chortis Block originates from a west-southwest position in the Pacific rather than attached to the Mexican margin. Additionally, this model proposes a high rate (200 km/20 Myr) of trench erosion along the Mexican margin just north of the present-day Chortis Block. In the Pacific model, the Chortis Block is being pushed eastward by the subduction movement of the Farallon and Cocos oceanic plates, and is finally captured by the Caribbean plate. In this study, the evolution of subduction along the Mexican and Central America margins is investigated numerically taking into account kinematic reconstruction of the Pacific model. The main goal is to investigate validity of the Pacific model for Chortis Block evolution by comparing our predicted slab geometries with present-day tomographic images. The kinematic 3D numerical models are integrated 45 Myr in time and use a mantle reference frame for tectonic plate evolution of the study area. These models generate subducting plate geometries that are only partial consistent with what is observed from seismic tomography, they reproduce realistic slab geometries only for the region located beneath the North America Plate, where the Cocos slab is flat at shallow depths and also stagnates along the mantle transition zone. On the other hand, for the region located beneath the Chortis Block the slab penetrates vertically through the upper mantle and even curls back in the upper portion of the lower mantle, a geometry that is inconsistent with seismic tomography images which shows the presence of east-dipping slab that tends to stagnate at 1000 km depth. This study indicate that the Pacific model is not an appropriate hypothesis for explaining the Chortis Block origin, and future studies should focus on alternative tectonic models. • Modeling the Middle America subduction tailored to the Pacific model using kinematic 3D numerical models. • Testing the Pacific model hypothesis for explaining the Chortis Block origin. • Understanding the global controlling factors for subduction in this region. • Comparing subduction models with tomography images for the study area. • Comparing upper mantle flow patterns above the slab with seismic anisotropy studies. [ABSTRACT FROM AUTHOR]

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different. [ABSTRACT FROM AUTHOR]

Abstract: Recently, the Colombian party system has undergone dramatic change as ‘new’ political actors have become increasingly successful in national legislative elections. This paper conducts a two-fold analysis of changes in the Colombian party system. First, the study explores several factors, linked to electoral and registration reforms, which have led to the increased share of seats awarded to ‘new’ actors, from 1982 to 1998. Next, it examines the substantive effects of those changes for the kinds of policies and issues pursued by new and traditional parties in the national legislature. The findings suggest that in spite of notable changes that have lead to a proliferation of new actors, the Colombian political system has not been radically changed. In other words, it is not evident that reforms have produced truly distinct political alternatives in Colombia. I argue that this is largely a function of incomplete reforms that allowed for the ‘legalization’ of traditional party factions and the persistence of electoral incentives that do not encourage distinct forms of behavior. [Copyright &y& Elsevier]

The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction (∼50%) in 22Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics. [ABSTRACT FROM AUTHOR]

• Prenatal diets containing high folic acid and low vitamin B12 (HFLB12) affect hepatic fatty acids and lipid metabolism of dams and their offspring. • In liver of dams, the HFHB12 diet decreased amount of all fatty acids: SFA; MUFA, PUFA and LC-PUFA; these last by a mechanism involving a decreased activity of both desaturases enzymes (Δ−5 and Δ−6). • In liver of offspring at 60 days old, the effects were opposite than their mothers, being more noticeable in females. Micronutrients (folates and vitamin B12) and long chain polyunsaturated fatty acids (LC-PUFAs) are linked through the one carbon cycle. We studied the effects of pre and postnatal high FA/low B12 diets (HFLB12) on hepatic fatty acid metabolism. Pregnant C57BL/6 mice were divided in two groups: control (2 mg folic acid: FA/25 µg vitamin B12/Kg food) and HFLB12 diets (8 mg FA/5 µg vitamin B12/Kg food). Offspring continued on the same diets until 60 days old. We determined hepatic fatty acid profile in dams and offspring and the expression of PPARα, Cpt-1, Acox-1 and Fas and the enzymatic activity of desaturases, all involved in lipid metabolism. In liver of dams, the HFHB12 diet decreased total fatty acids and desaturase activities; in offspring, effects were opposite, being more noticeable in females. Prenatal and postnatal unbalanced folic acid/B12 diets play a crucial role in regulating genes and enzymes involved in lipid metabolism in liver of dams and their offspring in adulthood. [ABSTRACT FROM AUTHOR]

Chagas disease (CHD) is the highest economic burden parasitosis worldwide and the most important cardiac infection, without therapeutic alternatives to halt or reverse its progression. In CHD-experimental models, antioxidant and anti-inflammatory compounds have demonstrated therapeutic potential in cardiac dysfunction. Theobroma cacao polyphenols are potent natural antioxidants with cardioprotective and anti-inflammatory action, which are susceptible to degradation, requiring technological approaches to guarantee their protection, stability, and controlled release. Here, 21 cocoa polyphenol-rich microencapsulates were produced by spray-drying and freeze-drying techniques using two wall materials (maltodextrin and gum arabic). Chemical (total and individual phenolic content and antioxidant activity), structural (morphology), and biological parameters (cytotoxicity, trypanocidal, antioxidant, and immunomodulatory activities) were assessed to determine the most efficient microencapsulation conditions on Trypanosoma cruzi -infected myocardioblast and macrophage cells. Significant antiproliferative properties against infected cells (superior to benznidazole) were found in two microencapsulates which also exhibited cardioprotective properties against oxidative stress, inflammation, and cell death. [ABSTRACT FROM AUTHOR]

The renal Na+:Cl- cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl- influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl- efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr53 and Thr58. Elimination of these threonines together with serine residue Ser71 completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation. [ABSTRACT FROM AUTHOR]