Evidence of a structural and functional ammonium transporter RhBG·anion exchanger 1·ankyrin-G complex in kidney epithelial cells.

The renal ammonium transporter RhBG and anion exchanger 1 kAE1 colocalize in the basolateral domain of α-intercalated cells in the distal nephron. Although we have previously shown that RhBG is linked to the spectrin-based skeleton through ankyrin-G and that its NH3 transport activity is dependent on this association, there is no evidence for an interaction of kAE1 with this adaptor protein. We report here that the kAE1 cytoplasmic N terminus actually binds to ankyrin-G, both in yeast two-hybrid analysis and by coimmunoprecipitation in situ in HEK293 cells expressing recombinant kAE1. A site-directed mutagenesis study allowed the identification of three dispersed regions on kAE1 molecule linking the third and fourth repeat domains of ankyrin-G. One secondary docking site corresponds to a major interacting loop of the erythroid anion exchanger 1 (eAE1) with ankyrin-R, whereas the main binding region of kAE1 does not encompass any eAE1 determinant. Stopped flow spectrofluorometry analysis of recombinant HEK293 cells revealed that the Cl(-)/HCO3 (-) exchange activity of a kAE1 protein mutated on the ankyrin-G binding site was abolished. This disruption impaired plasma membrane expression of kAE1 leading to total retention on cytoplasmic structures in polarized epithelial Madin-Darby canine kidney cell transfectants. kAE1 also directly interacts with RhBG without affecting its surface expression and NH3 transport function. This is the first description of a structural and functional RhBG·kAE1·ankyrin-G complex at the plasma membrane of kidney epithelial cells, comparable with the well known Rh·eAE1·ankyrin-R complex in the red blood cell membrane. This renal complex could participate in the regulation of acid-base homeostasis.

Rapid Cl⁻/HCO⁻3exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells.

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters.

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4+ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4+ and MeA+ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH4+, or cotransports NH3/H+, RhCG mediates electroneutral NH3 transport.

Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1).

Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3- and Cl- exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl-/HCO3- exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3- and outward Cl- gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3- for stomatin-deficient patients. Similarly, kinetics of Cl- efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells.

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.

Tuberculosis en América Latina y el Caribe: reflexiones desde la bioética / Tuberculosis in Latin America and the Caribbean: Reflections from Bioethics / Tuberculose na América Latina e no Caribe: reflexões da bioética

Resumen El objetivo de este artículo es analizar las condiciones de acceso a servicios de salud de las personas con tuberculosis en América Latina y el Caribe (ALC), reflexionando desde la bioética sobre los aspectos de salud pública implicados. Se realizó una revisión documental del contexto de la tuberculosis en ALC con base en datos epidemiológicos. Los resultados se analizaron a partir de su relación con los determinantes sociales de la salud, los principios éticos que pautan la práctica médica y la responsabilidad social de los actores de salud. La tuberculosis es un problema de salud pública acuciante en la región, debido a su impacto familiar, comunitario, social, económico y sanitario, que afecta principalmente a personas y poblaciones vulneradas. En ALC la tuberculosis constituye un serio problema ético y de salud pública que causa un significativo número de muertes, discapacidad e incremento de la pobreza. La comprensión de su responsabilidad social por parte de los actores de salud es imperativa para cumplir con el derecho a servicios de salud de calidad, que aseguren un diagnóstico oportuno y un tratamiento completo de la enfermedad, y que guarden los principios de justicia, no discriminación y dignidad de los/las enfermos/as, para lo cual es importante que las estrategias nacionales de control de la tuberculosis incluyan cambios en los determinantes sociales de la enfermedad, así como el respeto de la etnia, cultura, lengua e identidad de los pacientes.

Abstract The objective of this article is to analyze the conditions of access to health services by people with tuberculosis in Latin America and the Caribbean (LAC), reflecting on the public health aspects involved from a bioethical perspective. A literature review of the context of tuberculosis in LAC based on epidemiological data was performed. The results were analyzed from its relationship with the social determinants of health and the ethical principles that guide medical practice. Tuberculosis is a pressing public health problem in the region because of its family, social, economic and health impact. It mainly affects vulnerable individuals and populations. Health services violate ethical principles. Tuberculosis is a serious ethics and public health problem in the region that causes death, disability and increased poverty. It is imperative to ensure the right to health services and to understand the individual and public health consequences of non-adherence to treatment. It is important that national tuberculosis control strategies include principles of dignity and non-discrimination of the sick, changes in the social determinants of the disease, and respect for the ethnicity, language culture and identity of patients.

Resumo O objetivo deste artigo é analisar as condições de acesso aos serviços de saúde para pessoas com tuberculose na América Latina e no Caribe (ALC), refletindo a partir da bioética sobre os aspectos de saúde pública envolvidos. Uma revisão documental do contexto da tuberculose na ALC foi realizada com base em dados epidemiológicos. Os resultados foram analisados com base em sua relação com os determinantes sociais da saúde, os princípios éticos que norteiam a prática médica e a responsabilidade social dos atores da saúde. A tuberculose é um problema urgente de saúde pública na região, devido ao seu impacto familiar, comunitário, social, econômico e sanitário, que afeta principalmente pessoas e populações vulneráveis. Na ALC, a tuberculose é um grave problema ético e de saúde pública que causa um número significativo de mortes, deficiências e aumento da pobreza. É imperativo que os agentes de saúde compreendam a sua responsabilidade social para que, dessa forma, seja possível garantir o direito a serviços de saúde de qualidade, que assegurem o diagnóstico oportuno e o tratamento completo da doença, e que defendam os princípios de justiça, não-discriminação e a dignidade dos/das pacientes, para a qual é importante que as estratégias nacionais de controle da tuberculose incluam mudanças nos determinantes sociais da doença, bem como o respeito à etnia, cultura, língua e identidade dos pacientes.

Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG.

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells.

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.

Two nonerythroid homologs of the blood group Rh proteins, RhCG and RhBG, which share homologies with specific ammonia transporters in primitive organisms and plants, could represent members of a new family of proteins involved in ammonia transport in the mammalian kidney. Consistent with this hypothesis, the expression of RhCG was recently reported at the apical pole of all connecting tubule (CNT) cells as well as in intercalated cells of collecting duct (CD). To assess the localization along the nephron of RhBG, polyclonal antibodies against the Rh type B glycoprotein were generated. In immunoblot experiments, a specific polypeptide of Mr approximately 50 kD was detected in rat kidney cortex and in outer and inner medulla membrane fractions. Immunocytochemical studies revealed RhBG expression in distal nephron segments within the cortical labyrinth, medullary rays, and outer and inner medulla. RhBG expression was restricted to the basolateral membrane of epithelial cells. The same localization was observed in rat and mouse kidney. RT-PCR analysis on microdissected rat nephron segments confirmed that RhBG mRNAs were chiefly expressed in CNT and cortical and outer medullary CD. Double immunostaining with RhCG demonstrated that RhBG and RhCG were coexpressed in the same cells, but with a basolateral and apical localization, respectively. In conclusion, RhBG and RhCG are present in a major site of ammonia secretion in the kidney, i.e., the CNT and CD, in agreement with their putative role in ammonium transport.

Neumomediastino espontáneo: revisión de seis casos en el Hospital del Niño / Spontaneous pneumomediastinum: review of 6 cases in the Hospital del Niño

Se revisaron los seis casos de neumomediatismo espontáneo (NME) ocurridos en el Hospital del Niño desde 1981; encontrando una buena evolución con curación en tres casos y, tres fallecieron por agravamiento de la enfermedad básica. La revisión de la literatura confirma el tratamiento conservador del NME, pero en algunos casos se hace necesaria la faciotomía del cuello y/o la traqueostomía

Evolución de la tuberculosis en el Hospital del Niño en 10 años / Course of tuberculosis at the Hospital del Niño in 10 years

Se hizo la revisión de los casos de Tuberculosis hospitalizados durante 10 años en el Hospital del Niño (1975-1984) encontrándose una pobre fluctuación de las tasas anuales, con un predominio de pacientes de 1 a 5 años. Todavía en 1983 se reportaron casos de tuberculosis miliar y de meningitis tuberculosa pero desde este mismo año no hubo ninguna defunción por tuberculosis. Se destacó la importancia de la B.C.G. y se analizaron los diferentes métodos diangósticos de algunas formas de tuberculosis

Inhaloterapia: evaluación preliminar de un año de atención en el Hospital del Niño / Inhalation therapy: a year of care evaluation at the Hospital del Niño

Se hizo la evaluación preliminar de un año de atención en el Servicio de Inhaloterapia del Hospital del Niño. El 72 (por ciento) de los niños atendidos fueron menores de 5 años y el 66 (por ciento) de los pacientes se controlaron ambulatoriamente. Las principales causas de morbilidad atendidas fueron el asma y la bronquitis. Los corregimientos más afectados fueron aquellos de mayor contaminación ambiental

Infecciones respiratorias bajas: algunas consideraciones / Infections respiratory fall: some considerations

Se revisaron los expedientes de pacientes con diagnóstico de infecciones respiratorias bajas, correspondientes al primer semestre de 1984. Se estudian cuales fueron los factores clínicos, evolución, tratamiento y complicaciones de dichas infecciones

Neumonías por mycoplasma pneumoniae / Mycoplasma Pneumoniae Pneumonias

Se presenta el caso de una niña de ocho meses de edad, enviada de un hospital de provincia después de varios tratamientos con antibióticos y en la cual se diagnosticó M. pneumoniae. La niña mejoró con Eritromicina de 40 mg/kg/vía oral. Se compara luego el inicio de las infecciones respiratorias agudas por M. pneumoniae o por otra etiología, se encontró una diferencia significativa con una presentación más aguda en las últimas

Broncoscopía y anestesia en pediatría : estudio comparativo en el Hospital del Niño durante el año 1989 / Bronchoscopy and Anesthesia in Pediatrics, Comparative Study at the Hospital del Niño in 1989

Se realizó el estudio comparativo de 58 niños de 0 a 15 años sometidos a broncoscopia con anestesia general y 42 niños de 0 a 15 años sometidos a brocoscopia con anestesia local. Se presentaron 11 complicaciones en el primer grupo y 3 complicaciones en el segundo. En el primer grupo las complicaciones fueron : 3 casos con arritmias cardíacas, 5 con extracción de un diente, 2 con broncoespasmo, 1 con broncoaspiración de comida. Con anestesia local las complicaciones fueron : 1 caso con laringoespasmo, 1 con tos espasmódica, 1 con extracción de un diente. Todas las complicaciones fueron resueltas inmediatamente. Se concluye que la anestesia local aún con la broncoscopia rígida produce menos complicaciones que la anestesia general

Prevalencia de la infección por Paracoccidioides brasiliensis en niños panameños: informe preliminar / The prevalence of Paracoccidioides brasiliensis in panamanian children: preliminary report

Mediante la aplicación intracutánea de 50 microgramos de paracoccidioidina en 110 niños demostramos la prevalencia de infección debida a la Paracoccidioides brasiliensis en 14 (12.73%) niños. Los varones se infectaron más frecuentemente que las mujeres, en proporción de 1.45:1. El niño de menor edad tenía 2 años. Observamos una relación directa entre la edad y la frecuencia de la infección (a mayor edad aumenta el número de niños con reacción positiva); siempre más frecuente en el varón. El 93% (13 de 14) de los niños infectados, vive y se desarrolla en ambiente rural

Tumores torácicos en Niños: Hospital del Niño 1980-1986 / Thoracic tumors in children: Hospital del Niño 1980-1986

Se revisaron los expedientes clínicos de 19 pacientes con diagnóstico de tumor torácico en el tiempo transcurrido desde 1980 a 1986. Se encontró que estos tumores representaban 2% de todos los tumores sólidos diagnosticados en este lapso de tiempo y había un predominio de los linfomas No-Hodgkin en 35% y sólo 10% de tumor intrapulmonar (Hamartoma). Clínicamente no se encontró diferencia entre los tumores primarios y metatásicos, pero en más de 60% de los casos el diagnóstico se realizó cuando ya había signos de compresión mediastinal; la citología pleural fue positiva en 80% de los casos con derrame pleural. La mortalidad fue de 1/3 para los tumores primarios y de 1/2 para los metastásicos, la causa inmediata en ambos casos fue la insuficiencia respiratória y la principal el linfoma No-Hodgkin. Además se reportó 60% de deserción temprana. La incidencia total de tumores sólidos fue de 7-10/10**5 niños para toda la República de Panamá

Tuberculoma intracraneal: Tratamiento médico y quirúrgico: Reporte de dos casos / Intracranial tuberculoma: Medical and surgical treatment: Report of two cases

Los autores presentan dos pacientes menores de quince años de edad que fueron atendidos en el Hospital del Niño, por presentar tuberculomas intracraneales. Uno con excelente respuesta al tratamiento antituberculoso y el otro tratado quirúrgicamente. La tomografía axial computarizada del cerebro, demostró la presencia de cuatro tuberculomas supratentoriales. Tres en el primer caso y uno en los ganglios basales del segundo caso. El diagnóstico se efectuó con las bases clínicas, radiológicas y terapeúticas

Evaluacion de la unidad de terapia intensiva del Hospital del Niño durante 1986

Se analizarón 190 casos atendidos en la Unidad de Terapia Intensiva del Hospital del Niño, desde el 1/1/86 hasta del 31/12/86. Se utilizó el Sistema de Clasificación Clínica (SCC) y el Sistema de Puntuación de la Intervención Terapéutica (TISS) para evaluar la severidad de los casos y los cuidados prestados a los pacientes. De los 190 pacientes admitidos 33 (23%) pertenecían al grupo de 1 a 11 meses, 57 (47%) al de 1 a 4 años y 32 (23%) al de 10 a 15 años. La mortalidad global fue de 24.4% (47 casos) siendo el grupo más afectado el de menos de un año de edad, que representa el 55% del total de defuncicones. Los sistemas más afectados fueron: respiratório (27.7%), neurológico (23%) y gastrointestinal (10%). El TISS promedio de los pacientes vivos fue de 11.6, mientras que fue de 24.7 en los pacientes que fallecieron. El sistema de puntuación TISS y el de Clasificación Clínica nos permiten evaluar la gravedad del pacietne admitido a la unidad, la terapia aplicada y los resultados en forma objetiva

Estado actual de la resistencia a los fármacos tuberculosos en el Hospital del Niño de Panamá / Current status of resistance to drugs against tuberculosos in the Hospital del Niño de Panamá

En un estudio retrospectivo en el Hospital del Niño, sobre 50 casos de tuberculosis diagnosticados entre 1991 y 1994, encontramos que un (38 por ciento) de los pacientes presentaron un cultivo positivo por Mycobacterium tuberculosis, que no hubo resistencia a los fármacos usados y que la curación clínica y radiográfica fue del (90 por ciento) cuando se utilizó el esquema de dos meses con isoniacida, rifampicina; sin embargo, un estudio realizado en Panamá en 359 pacientes adultos, dos años antes, se halló una resistencia secundaria del (7 por ciento). Así que, es de esperar que se desarrolle, en los próximos años, una resistencia en los niños si no se refuerza la vigilancia epidemiológica de los adultos