Effects of mineralocorticoid and K+ concentration on K+ secretion and ROMK channel expression in a mouse cortical collecting duct cell line.

The cortical collecting duct (CCD) plays a key role in regulated K(+) secretion, which is mediated mainly through renal outer medullary K(+) (ROMK) channels located in the apical membrane. However, the mechanisms of the regulation of urinary K(+) excretion with regard to K(+) balance are not well known. We took advantage of a recently established mouse CCD cell line (mCCD(cl1)) to investigate the regulation of K(+) secretion by mineralocorticoid and K(+) concentration. We show that this cell line expresses ROMK mRNA and a barium-sensitive K(+) conductance in its apical membrane. As this conductance is sensitive to tertiapin-Q, with an apparent affinity of 6 nM, and to intracellular acidification, it is probably mediated by ROMK. Overnight exposure to 100 nM aldosterone did not significantly change the K(+) conductance, while it increased the amiloride-sensitive Na(+) transport. Overnight exposure to a high K(+) (7 mM) concentration produced a small but significant increase in the apical membrane barium-sensitive K(+) conductance. The mRNA levels of all ROMK isoforms measured by qRT-PCR were not changed by altering the basolateral K(+) concentration but were decreased by 15-45% upon treatment with aldosterone (0.3 or 300 nM for 1 and 3 h). The paradoxical response of ROMK expression to aldosterone could possibly work as a preventative mechanism to avoid excessive K(+) loss which would otherwise result from the increased electrogenic Na(+) transport and associated depolarization of the apical membrane in the CCD. In conclusion, mCCD(cl1) cells demonstrate a significant K(+) secretion, probably mediated by ROMK, which is not stimulated by aldosterone but increased by overnight exposure to a high K(+) concentration.

Las comunidades benticas entre los 03º y 09º S en mayo de 1995 (Cr. BIC SNP-1, 9505-06)

Estudio de la estructura de las comunidades bentónicas sublitorales de fondo blando mediante el análisis de 11 muestras de draga colectadas entre los 03°38 ' y 09°16 'S, entre el 12 y 19 de mayo de 1995. Se determinó la biodiversidad a nivel de especies de macrobentos para las subáreas A, B, C, D. Se evaluó el nivel de similaridad entre las 11 estaciones analizadas a nivel de grupos taxonómicos (Polychaeta, Crustacea, Mollusca, Nemertinea y otros), así como la relación entre el nivel de estrés de las comunidades de las subáreas latitudinales A, B, C, D y la agremiación en el grupo dominante (Polychaeta). Fueron diferenciadas un total de 73 especies macrobénticas de las cuales el 64,38% pertenece a la clase Polychaeta. El patrón de distribución de las biomasas totales del macrobentos es más simple que el de las abundancias, con una tendencia a la ubicación de los valores máximos al norte de los 04° S. Se comprueba una relación inversa entre el grado de agremiación y el nivel de estrés medido con el índice SEP.

Características del subsistema demersal durante el crucero de evaluación del recurso merluza (Cr. BIC SNP-1, 9505-06)

Describe la composición por especies de las capturas obtenidas durante la etapa de evaluación del crucero BIC SNP-1 9505-06, entre el 10 de mayo al 13 de junio de 1995, comprendiendo desde Huarmey hasta Puerto Pizarro. Se capturaron 80 especies entre peces (64), crustáceos (10), moluscos (4), equinodermos (1). La mayor diversidad de especies se encontró al norte de los 6°S (subáreas A, B, C, D); se destaca una disminución hacia el sur de este paralelo.

Molecular epidemiology and evolution of HIV-1 in Portugal and portuguese speaking african countries

The aims of this thesis were to better characterize HIV-1 diversity in Portugal, Angola, Mozambique and Cape Verde and to investigate the origin and epidemiological history of HIV-1 in these countries. The impact of these issues in diagnosis, disease progression and susceptibility to ARV therapy was also investigated. Finally, the nature, dynamics and prevalence of transmitted drug resistance (TDR) was determined in untreated HIV-1 infected patients. In Angola, practically all HIV-1 genetic forms were found, including almost all subtypes, untypable (U) strains, CRFs and URFs. Recombinants (first and second generation) were present in 47.1% of the patients. HIV/AIDS epidemic in Angola probably started in 1961, the major cause being the independence war, subsequently spreading to Portugal. In Maputo, 81% of the patients were infected with subtype C viruses. Subtype G, U and recombinants such as CRF37_cpx, were also present. The results suggest that HIV-1 epidemic in Mozambique is evolving rapidly in genetic complexity. In Cape Verde, where HIV-1 and HIV-2 co-circulate, subtype G is the prevailed subtype. Subtypes B, C, F1, U, CRF02_AG and other recombinant strains were also found. HIV-2 isolates belonged to group A, some being closely related to the original ROD isolate. In all three countries numerous new polymorphisms were identified in the RT and PR of HIV-1 viruses. Mutations conferring resistance to the NRTIs or NNRTIs were found in isolates from 2 (2%) patients from Angola, 4 (6%) from Mozambique and 3 (12%) from Cape Verde. None of the isolates containing TDR mutations would be fully sensitive to the standard first-line therapeutic regimens used in these countries. Close surveillance in treated and untreated populations will be crucial to prevent further transmission of drug resistant strains and maximize the efficacy of ARV therapy. In Portugal, investigation of a seronegative case infection with rapid progression to AIDS and death revealed that the patient was infected with a CRF14_BG-like R5-tropic strain selectively transmitted by his seropositive sexual partner. The results suggest a massive infection with a highly aggressive CRF14_BG like strain and/or the presence of an unidentified immunological problem that prevented the formation of HIV-1-specific antibodies. Near full-length genomic sequences obtained from three unrelated patients enabled the first molecular and phylogenomic characterization of CRF14_BG from Portugal; all sequences were strongly related with CRF14_BG Spanish isolates. The mean date of origin of CRF14_BG was estimated to be 1992. We propose that CRF14_BG emerged in Portugal in the early 1990s, spread to Spain in late 1990s as a consequence of IDUs migration and then to the rest of Europe. Most CRF14_BG strains were predicted to use CXCR4 and were associated with rapid CD4 depletion and disease progression. Finally, we provide evidence suggesting that the X4 tropism of CRF14_BG may have resulted from convergent evolution of the V3 loop possibly driven by an effective escape from neutralizing antibody response.

Harmonia axyridis: a threat to Brazilian Coccinellidae?

Harmonia axyridis (Pallas) (Coleoptera, Coccinellidae) is a polyphagous Asian species, well-known as a classical biological control agent of aphids around the world, introduced probably accidentally in Brazil, sampled for the first time in 2002. It is an important intraguild predator, competing for food with native coccinellids. It was studied H. axyridis alimentary sources and host plants, its abundance compared with native and established species, the influence of abiotic factors and the seasons over the abundance of H. axyridis throughout one year, and discussed the mechanisms which influence the displacement of species. Harmonia axyridis was found in 38 plant species, among them 20 were new records, feeding on 20 aphid species, eight of them new alimentary records. Between 2006/2007, eight Coccinellidae species were collected and H. axyridis was the most abundant (91.23%). Harmonia axyridis peak of abundance occurred in August and September 2007, probably influenced by the temperature and food availability. From 1999 to 2007 a reduction and variation in the diversity of collected species of Coccinellidae were observed with the predominance of H. axyridis, which may indicate their displacement.

Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.

The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

Comparative phylogeography of mutualists and the effect of the host on the genetic structure of its partners.

Whether or not species participating in specialized and obligate interactions display similar and simultaneous demographic variations at the intraspecific level remains an open question in phylogeography. In the present study, we used the mutualistic nursery pollination occurring between the European globeflower Trollius europaeus and its specialized pollinators in the genus Chiastocheta as a case study. Explicitly, we investigated if the phylogeographies of the pollinating flies are significantly different from the expectation under a scenario of plant-insect congruence. Based on a large-scale sampling, we first used mitochondrial data to infer the phylogeographical histories of each fly species. Then, we defined phylogeographical scenarios of congruence with the plant history, and used maximum likelihood and Bayesian approaches to test for plant-insect phylogeographical congruence for the three Chiastocheta species. We show that the phylogeographical histories of the three fly species differ. Only Chiastocheta lophota and Chiastocheta dentifera display strong spatial genetic structures, which do not appear to be statistically different from those expected under scenarios of phylogeographical congruence with the plant. The results of the present study indicate that the fly species responded in independent and different ways to shared evolutionary forces, displaying varying levels of congruence with the plant genetic structure

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Respuesta selectiva de la red de arrastre del fondo tipo 450/120 empleada en el estudio de recursos demersales a inicios del invierno 1998. Crucero BIC José Olaya Balandra 9806-07 de puerto Pizarro a Huarmey

Estudia la respuesta selectiva de la red de arrastre de fondo tipo 450/120, utilizada en la toma de muestras durante el Crucero BIC José Olaya Balandra 9806-07 empleando el método de copo cubierto, con un tamaño de malla de 90 mm. (Polipropileno PP). Se obtuvieron las ojivas de selección mediante el método de la curva logística, para la zona de toda la extensión que abarcó el crucero desde las subáreas A, B, C, D, E, F, G y H (3°30 'S - 11°00 'S), presentando una L50%=23,68 cm. y curvas de selectividad parciales de las subáreas A, B, C y D, E, F, G y H con una L50%=25,45 cm. y L50%=23,68 cm. respectivamente. Se encontró un factor de selección general de (FS)=2,63. Los resultados fueron mayores que en el experimento modelo de selectividad con red de arrastre de fondo, realizado en invierno 1997.

Caracterización especiológica bentodemersal durante el crucero de investigación del recurso merluza BIC Jose Olaya Balandra 9901

Los lances de pesca se efectuaron entre 96 y 560 m de profundidad, del 7 al 26 de enero de 1999, frente al área entre Huarmey (10°5 'S) y Puerto Pizarro (3°29 'S). Se registraron 168 especies: 97 peces y 71 invertebrados (35 crustáceos, 19 moluscos, 8 equinodermos, 3 poliquetos y 6 cnidarios). La captura total fue de 19.962,13 kg, de los cuales 18.041,53 kg (90,38%) correspondieron a peces; 93,88 kg (0.47%) a crustáceos; 11,65 kg (0,06%) a moluscos; 7,69 kg (0,04%) a equinodermos; 0,64 kg (0,03%) a poliquetos y 1.806,74 kg (9,05%) a cnidarios. El falso volador Prionotus stephanophrys destacó por su abundancia y frecuencia, habiéndose capturado 6.755,26 kg (33,8% del volumen total), seguido por la merluza Merluccius gayi peruanus con 4.823,99 kg (24,2%) y la cabrilla Paralabrax humeralis con 4.581,23 kg (23,0%). Se analiza también la distribución latitudinal y batimétrica de la fauna registrada, y se presentan las tallas de tres especies de peces (anguila común Ophichthus pacifici, congrio negro Cherublemma emmelas y el pez guadaña Hoplostethus pacificus) capturadas durante cinco operaciones de pesca. La diversidad específica y equitabilidad referidas a la merluza son objeto de análisis, y se determina que son significativamente bajas en el estrato I de la mayoría de subáreas en estudio, donde alcanzan valores hasta de cero, a excepción de la subárea F, mientras que en los estratos II y III de las subáreas A, B, C, D y F alcanzaron valores significativos.

Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors.

Receptor activity modifying proteins RAMP1, RAMP2, and RAMP3 are responsible for defining affinity to ligands of the calcitonin receptor-like receptor (CRLR). It has also been proposed that receptor activity-modifying proteins (RAMP) are molecular chaperones required for CRLR transport to the cell surface. Here, we have studied the respective roles of CRLR and RAMP in transporting CRLR/RAMP heterodimers to the plasma membrane by using a highly specific binding assay that allows quantitative detection of cell surface-expressed CRLR or RAMP in the Xenopus oocytes expression system. We show that: (i) heterodimer assembly is not a prerequisite for efficient cell surface expression of CRLR, (ii) N-glycosylated RAMP2 and RAMP3 are expressed at the cell surface and their transport to the plasma membrane requires N-glycans, (iii) RAMP1 is not N-glycosylated and is transported to the plasma membrane only upon formation of heterodimers with CRLR, and (iv) introduction of N-glycosylation sites in the RAMP1 sequence (D58N/G60S, Y71N, and K103N/P105S) allows cell surface expression of these mutants at levels similar to that of wild-type RAMP1 co-expressed with CRLR. Our data argue against a chaperone function for RAMP and identify the role of N-glycosylation in targeting these molecules to the cell surface.

Recurrent deletions and reciprocal duplications of 10q11.21q11.23 including CHAT and SLC18A3 are likely mediated by complex low-copy repeats.

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.

Selectividad de la red Granton 400x120 mm PA en la evaluación de merluza peruana. Otoño 2002

En el crucero BIC Olaya 0205 (Callao-Puerto Pizarro), se monitoreó la selectividad de la red de arrastre de fondo Granton tipo 400x120 mm PA, empleando copo (90 mm) y sobrecopo (13 mm). La probabilidad de captura al 50%, en la subárea A fue de 4,52cm; en la B, de 25,46 cm y en la C de 26,14 cm. El L50% total de las subáreas A, B, C fue 25,15 cm, con un factor de selección 0,28 y un rango de selección de 8,55 cm.

Dinâmica da agregação induzida pelo uso de plantas de inverno para cobertura do solo

O impacto de plantas de inverno sobre a estabilidade estrutural do solo, antecedendo a cultura do milho sob plantio direto, foi avaliado em Podzólico Vermelho-Amarelo. O experimento foi realizado no campus da Universidade Federal de Santa Maria, de maio de 1991 a maio de 1992. Os tratamentos utilizados constaram de: chícharo (Lathyrus sativus L.), tremoço azul (Lupinus angustifolius L.), ervilhaca (Vicia sativa L.), aveia preta (Avena strigosa Schieb) e pousio invernal. Em cada parcela, foram coletadas mensalmente, desde a implantação das plantas de inverno até a colheita do milho, totalizando treze coletas, amostras de solo parcialmente deformadas e indeformadas para as determinações. Foram usadas duas subamostras na profundidade de 0-5 cm, com pá de corte, compondo uma amostra para análise de agregados e carbono orgânico e cinco subamostras, na mesma profundidade, com uso de cilindro volumétrico, compondo amostra para análise da atividade microbiana e umidade do solo. As plantas de cobertura induziram variação temporal da estabilidade dos agregados no período de estudo. A aveia preta atingiu maiores valores de estabilidade estrutural durante o ciclo das culturas de inverno, enquanto o tremoço azul maiores valores durante o ciclo do milho. Isso pode ser atribuído ao sistema radicular da gramínea e à taxa de decomposição da leguminosa, criando ambiente favorável à agregação, pela ação de raízes, cobertura do solo, fornecimento de material orgânico e conservação da umidade favoráveis à ação de microrganismos. Tais fatores, provavelmente, favoreceram a formação e conservação dos agregados do solo.

Expertises croisées dans la dégénérescence maculaire liée à l'âge. Focus sur la physiopathologie, l'angiogenèse, les données pharmacologiques et cliniques [Review and expert opinion in age related macular degeneration. Focus on the pathophysiology, angiogenesis and pharmacological and clinical data].

Age related macular degeneration (AMD) is a pathological aging of the macula, brought about by the interaction of genetic and environmental factors. It induces geographic atrophy of the retina and/or choroidal neovascularization. In the latter, abnormal vessels develop from the choriocapillaris, with the involvement of VEGF (vascular endothelial growth factor). The VEGF family includes several factors, including VEGF-A, B, C, D, F and PlGF (placental growth factor). Their biological properties and their affinities to the VEGFR1, VEGFR2 and VEGFR3 receptors found on endothelial cells differ. Exudative AMD involves mainly VEGF-A and VEGF-R2. Anti-VEGF agents used in ophthalmology (ranibizumab, bevacizumab and aflibercept) are designed to primarily target this pathway. In vitro, all have sufficient affinity to their ligands. Their therapeutic efficacy must therefore be judged based on clinical criteria. In clinical practice, the minimum number of injections required for a satisfactory result appears to be comparable with all the three. The few available studies on therapeutic substitutions of anti-VEGF compounds suggest that some patients may benefit from substituting the anti-VEGF in cases of an unsatisfactory response to an initial molecule. Although local side effects, including increased risk of geographic atrophy, and systemic effects, including vascular accidents, have been suggested, these risks remain low, specially compared to the benefits of the treatment. Differences in safety between anti-VEGF are theoretically possible but unproven.