Efecto del entrenamiento de músculos abdominales sobre la función respiratoria en adolescentes sanos: Estudio piloto / Effect of abdominal muscle training on respiratory function in healthy adolescents: A preliminar study

The respiratory muscle training has been used to improve muscle strength and optimize the mechanism of cough. The aim of this study is to evaluate the effect of abdominal muscle training on respiratory muscle strength (MIP, MEP), peak expiratory flow (PEF) and peak cough flow (PCF) in healthy adolescents. The study design was quasi-experimental, variables of respiratory function were assessed before and after the muscle training protocol in a sample of sedentary healthy adolescents without gender restriction. The sample size calculated was 15 subjects. The training protocol consisted in 8 weeks of training divided into two stages (basic and advanced training plan) twice a week. Descriptive statistics were performed, tests for normality (Shapiro Wilk), U Mann Whitney test, Pearson coefficient and t-student test were used. Results are expressed as mean and its standard deviation. It was considered significant a p value < 0,05. Seventeen subjects (8 males and 9 females) entered to the study, all subjects performed the protocol and completed it without incidents. After completing the training protocol the sit-up test performance increased 21.7% (p = 0.0001), the MIP increase 16,5 cm H2O (17.1%) (p = 0.006), MEP increased 34.9 cm H2O (50,2 %) (p = 0.0001). Moreover, PEF increased 35.3 L/min (8.3%) (p = 0.003) and PCF increased 36.6 L/min (9,1%). There was no correlation between sit-up test performance and lung function variables. PEF showed only a moderate correlation with PCF (r = 0.6; p = 0.007) and MEP (r = 0.59; p = 0.01). We conclude that, in this sample, increases in respiratory muscle strength, PEF and PCF were observed after abdominal muscle training. No association between abdominal muscle strength and respiratory function variables was found before initiating the training protocol.

El entrenamiento muscular respiratorio ha sido utilizado para mejorar la fuerza de los músculos y optimizar el mecanismo de la tos. El objetivo de este estudio es evaluar el efecto del entrenamiento de músculos abdominales sobre la fuerza de los músculos respiratorios (Pimax, Pemax), flujo espiratorio máximo (FEM) y flujo máximo de tos (FMT) en adolescentes sanos. Este estudio es cuasi experimental, donde se evaluaron las variables de función respiratoria antes y después del protocolo de entrenamiento. La muestra estuvo integrada por adolescentes sanos sedentarios sin restricción de sexo. La muestra mínima estimada fue de 15 sujetos. El protocolo de entrenamiento consistió en 8 semanas de entrenamiento dividido en 2 etapas (plan básico y avanzado de ejercicios) dos veces por semana. Se realizó estadística descriptiva, pruebas de normalidad (Shapiro Wilk); se utilizó test U de Mann Whitney, coeficiente de correlación de Pearson y test t-student para muestras pareadas. Los resultados se expresan en promedios y desviación estándar. Se consideró significativo un valor de p < 0,05. Ingresaron al estudio 17 sujetos (8 hombres y 9 mujeres), todos los sujetos realizaron el protocolo completo. Posterior al protocolo los sujetos incrementaron en 21,7% el rendimiento del sit-up test (p=0,0001); la Pimax aumentó 16,5 cm H2O (+17,1%) (p=0.006), la Pemax aumentó 34,9 cm H2O (+50,2%) (p=0,0001). Por otra parte, el FEM aumentó 35,3 L/min (+8,3%) (p=0,003) y el FMT aumentó 36,6 L/min (+9,1%). La fuerza de músculos abdominales no muestra correlación con las variables de función respiratoria. Sólo FEM muestra correlación moderada con el FMT (r = 0,6; p = 0,007) y Pemax (r = 0,59; p = 0,01). Se concluye que, en la muestra estudiada, se observan incrementos en la fuerza muscular respiratoria, FEM y FMT luego de un protocolo de entrenamiento de músculos abdominales. No se observó asociación entre la fuerza muscular abdominal y las variables de función respiratoria antes de iniciado el protocolo.

Measles outbreak in Andalusia, Spain, January to August 2011.

On 7 January 2011, a six year-old child living in a Roma community near Seville, southern Spain, was hospitalised with measles. Contact tracing identified a probable index case with onset of symptoms on 20 December 2011 and several unreported cases among children under the age of 15 years in the same town. The outbreak initially spread in districts in the city of Seville with a high proportion of Roma residents, and later to other cities and towns in Andalusia. While some towns experienced wide spread of the disease with significant clusters of cases, most of the affected locations saw non-clustered cases or very few secondary cases. The outbreak resulted in 1,759 confirmed or probable cases of which 393 (19%) required hospitalisation. Measles virus of genotype D4 was diagnosed in more than half of the cases. Significant differences (p<0.0001) by age group were found between clustered and non-clustered cases. The highest proportion of clustered cases occurred in the age group of 5-14 yearolds, while the highest proportion of non-clustered cases was seen in those older than 29 years. The last confirmed case related to this outbreak was reported on 20 August 2011.

Sorting functions of the individual cytoplasmic domains of the G protein-coupled vasopressin V(2) receptor in Madin Darby canine kidney epithelial cells.

Previous studies have shown that the G protein-coupled human vasopressin V(2) receptor (V(2) receptor) is expressed predominantly in the basolateral membrane of Madin Darby canine kidney type II (MDCKII) epithelial cells at steady state. Here we have assessed the influence of the individual cytoplasmic domains of the V(2) receptor on polarized sorting in MDCKII cells. The second (ICL2) and third (ICL3) intracellular loops and the C-terminal tail were fused separately to a green fluorescent protein-tagged receptor fragment comprising the first transmembrane domain and flanking regions. We show that the ICL2 domain of the V(2) receptor alone promotes basolateral cell surface expression and thus seems to contain the basolateral sorting signal of the V(2) receptor. Fusion of the other cytoplasmic domains, however, does not lead to a randomized cell surface expression. The C-terminal tail of the V(2) receptor promotes apical targeting. Fusion of ICL3 leads to a receptor fragment that is retained in the endoplasmic reticulum (ER). The results are consistent with a model in which the V(2) receptor contains signals for both apical and basolateral cell surface expression, the latter being dominant. Furthermore, ICL3 may contain a RXR [corrected] ER retention signal, which is not accessible in the correctly folded full-length receptor but which is unmasked when ICL3 is fused alone.

Molecular and conformational features of a transport-relevant domain in the C-terminal tail of the vasopressin V(2) receptor.

We have previously shown a conserved glutamate/dileucine motif ((335)ELRSLL(340)) in the intracellular C terminus of the vasopressin V(2) receptor (V(2) receptor) to be essential for receptor transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The motif may represent a transport signal that is recognized by a component of ER to Golgi vesicles. Alternatively, it may be necessary for transport-competent receptor folding to pass the quality-control system of the ER. To assess these two possibilities, we constructed a receptor fragment that allows transport studies independent of full-length receptor folding. Transmembrane domains II-VII were deleted, thereby fusing the intracellular C terminus to the first cytoplasmic loop. The mutations that impaired transport of the full-length receptor were introduced, and receptor fragments were localized in transiently transfected HEK 293 cells. All mutant receptor fragments were detectable at the plasma membrane, demonstrating that the glutamate/dileucine motif does not function as a small, linear vesicular transport signal. Instead, our data strongly suggest that this motif is required for transport-competent folding of the full-length receptor. To assess the underlying conformational features, a three-dimensional homology model of the V(2) receptor was computed. Our model predicts that the glutamate/dileucine motif contributes to a U-like loop within the intracellular C terminus. Residue Leu(339) may be required for folding back the intracellular C terminus to residue Leu(62) of the first cytoplasmic loop. We characterized the naturally occurring L62P and DeltaL62-R64 mutations in the first cytoplasmic loop and show that they lead to transport-defective full-length V(2) receptors that are retained in the ER, consistent with the structure model.

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues.

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.

A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells.

Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.

Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells.

We have analyzed the polarized cell surface expression of the G protein-coupled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged receptors. Cell surface biotinylation assays with stably transfected filter-grown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter- and glass-grown MDCK cells stably transfected with a GFP-tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP-tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally show that microinjection of a plasmid encoding the GFP-tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein-coupled receptors. The methodology may be applicable to any cell type, even to tissue-derived, primary cultured cells; coinjection of transport-regulating compounds should also be possible.