Relação temporal entre apnéia obstrutiva e refluxo gastroesofágico em lactentes

O refluxo gastroesofágico (RGE) tem sido diagnosticado em uma elevada proporção de lactentes com apnéia e episódios de ALTE (apparent life threatening events) ou eventos com aparente risco de vida, entretanto, a relação entre eles per-manece pouco entendida. A proposta deste estudo é avaliar a coexistência de apnéia e refluxo gastroesofágico em um grupo de lactentes durante o sono e vigília, através da utilização da polissonografia e, pela inclusão de um canal para aferição do pH na porção distal do esôfago, determinar a relação temporal entre estes dois eventos e aferir a associação entre ALTE e RGE. Método: A população em estudo constituiu-se de lactentes com história clínica compatível com eventos sugestivos de RGE, além de alguma anormalidade do ponto de vista ventilatório, os quais realizaram polissonografia com a inclusão de um canal para registro contínuo do pH esofágico. Os estudos polissonográficos foram analisados individualmente, em três etapas assim constituídas: Etapa 1 - realização do escore dos eventos respiratórios e dos estágios de sono e vigília, com o pesquisador cego para a ocorrência de RGE; Etapa 2 - indicação do escore isolado dos eventos de RGE; Etapa 3 - reintrodução de canal do pH e estabelecimento da correlação temporal entre os dois eventos, levando-se em consideração o período compreendido entre um minuto antes da queda do pH abaixo de 4,0, até um minuto após a elevação do pH acima de 4,0. A relação entre a história clínica de ALTE e a ocorrência de RGE durante o estudo polissonográfico também foi aferida. A partir de dados publicados na literatura, foi calculada uma amostra de 88 indivíduos (lactentes). A amostra final constituiu-se de 89 pacientes. Resultados: Quarenta pacientes apresentaram RGE e quarenta e nove não. Ambos os grupos tinham composição semelhante quanto à idade, peso, duração total do estudo polissonográfico, duração do tempo total de sono durante o estudo e história de ALTE. Ocorreu uma média de 2,82 episódios de RGE por paciente (1-20 episódios/ paciente). Os episódios de RGE foram mais freqüentes e longos durante o sono que durante os períodos de vigília. O número de eventos obstrutivos/minuto de sono (densidade de apnéias) foi significativamente maior durante os períodos de RGE (mediana 0,4), se comparados aos períodos sem RGE (mediana 0,2), p = 0,002. Em relação às apnéias centrais, não houve diferença estatisticamente significativa, p = 0, 039. Em 62% dos períodos de RGE, houve associação temporal com eventos obstrutivos, dos quais a apnéia obstrutiva precedeu o RGE em 67% dos casos. A correlação entre história de ALTE e a presença de RGE não foi estatisticamente significativa. Conclusões: Os resultados sugerem que a apnéia obstrutiva em lacten-tes está freqüentemente associada com RGE do ponto de vista de temporalidade, e é mais provável que ela ocorra precedendo um episódio de refluxo. Especula-se que a apnéia obstrutiva no lactente possa, em algumas circunstâncias, ser a causa e não a conseqüência do RGE.

Nonacid reflux episodes reaching the pharynx are important factors associated with cough

BACKGROUND: Gastroesophageal reflux is implicated in the pathogenesis of asthma and chronic cough. To date most studies have focused on acid reflux measured by pH below the upper esophageal sphincter (UES). The aim of this study was to assess the relationship between cough and reflux through the UES into the pharynx. METHODS: Thirty-seven patients with asthma (19) and chronic cough (18) were recruited from the respiratory clinic. Reflux was monitored using a combined multichannel intraluminal impedance and pH probe by detecting (1) bolus reflux episodes within the esophagus and in the pharynx and (2) acidic reflux episodes within the esophagus and in the pharynx. All acid suppressive therapy was stopped for at least 7 days before the study. Demonstration of cough being linked to reflux was achieved using the symptom association probability (SAP). This was calculated using a 2-minute association window between symptoms and bolus entry into the esophagus. SAP was considered positive if >95%. RESULTS: A positive SAP for cough was noted in 7/26 patients reporting symptoms on the day of monitoring. Compared with SAP-negative patients, SAP-positive patients had both a greater number [median (interquartile range), 5(2 to 8) vs. 2(0 to 4), P<0.05] and a higher proportion of reflux episodes crossing the UES into the pharynx [25%(14% to 28%) vs. 7% (2% to 14%), P<0.02]. There was no difference in the number of reflux episodes or acid exposure time in the distal esophagus between SAP-positive and SAP-negative patients. Only 1% to 2% of episodes were detected by the pharyngeal pH sensor. CONCLUSIONS: Impedance detected pharyngeal reflux episodes are important factors in symptom production in cough patients.

Développement et caractérisation de dérivés dipyrrométhène pour des applications dans le domaine du photovoltaïque

Ce projet de recherche mené en collaboration industrielle avec St-Jean Photochimie Inc. / PCAS Canada vise le développement et la caractérisation de dérivés dipyrrométhène pour des applications dans le domaine du photovoltaïque. La quête du récoltage des photons se situant dans le proche-infrarouge a été au centre des modifications structurales explorées afin d’augmenter l’efficacité de conversion des cellules solaires de type organique et à pigments photosensibles. Trois familles de composés intégrant le motif dipyrrométhène ont été synthétisées et caractérisées du point de vue spectroscopique, électrochimique, structural ainsi que par modélisation moléculaire afin d’établir des relations structures-propriétés. La première famille comporte six azadipyrrométhènes au potentiel de coordination tétradentate sur des centres métalliques. Le développement d’une nouvelle voie synthétique asymétrique combinée à l’utilisation d’une voie symétrique classique ont permis d’obtenir l’ensemble des combinaisons de substituants possibles sur les aryles proximaux incluant les noyaux 2-hydroxyphényle, 2-méthoxyphényle et 2- pyridyle. La modulation du maximum d’absorption dans le rouge a pu être faite entre 598 et 619 nm. De même, la présence de groupements méthoxyle ou hydroxyle augmente l’absorption dans le violet (~410 nm) tel que démontré par modélisation. La caractérisation électrochimique a montré que les dérivés tétradentates étaient en général moins stables aux processus redox que leur contre-parti bidentate. La deuxième famille comporte dix dérivés BODIPY fusionnés de façon asymétrique en position [b]. L’aryle proximal a été modifié de façon systématique afin de mieux comprendre l’impact des substituents riches en électron et de la fusion de cycles aromatiques. De plus, ces dérivés ont été mis en relation avec une vaste série de composés analogues. Les résultats empiriques ont montré que les propriétés optoélectroniques de la plateforme sont régies par le degré de communication électronique entre l’aryle proximal, le pyrrole sur lequel il est attaché et le noyau indolique adjacent à ce dernier. Les maximums d’absorption dans le rouge sont modulables entre 547 et 628 nm et la fluorescence des composés se situe dans le proche- infrarouge. L’un des composé s’est révélé souhaitable pour une utilisation en photovoltaïque ainsi qu’à titre de sonde à pH. La troisième famille comporte cinq complexes neutres de RuII basés sur des polypyridines et portant un ligand azadipyrrométhène cyclométalé. Les composés ont montré une forte absorption de photons dans la région de 600 à 800 nm (rouge à proche- infrarouge) et qui a pu être étendue au-delà de 1100 nm dans le cas des dérivés portant un ligand terpyridine. L’analyse des propriétés optoélectroniques de façon empirique et théorique a montré un impact significatif de la cyclométalation et ouvert la voie pour leur étude en tant que photosensibilisateurs en OPV et en DSSC. La capacité d’un des complexes à photo-injecter un électron dans la bande de conduction du semi-conducteur TiO2 a été démontré en collaboration avec le groupe du Pr Gerald J. Meyer à University of North Carolina at Chapel Hill, premier pas vers une utilisation dans les cellules solaires à pigments photosensibles. La stabilité des complexes en solution s’est toutefois avérée problématique et des pistes de solutions sont suggérées basées sur les connaissances acquises dans le cadre de cette thèse.

Développement et caractérisation de dérivés dipyrrométhène pour des applications dans le domaine du photovoltaïque

Ce projet de recherche mené en collaboration industrielle avec St-Jean Photochimie Inc. / PCAS Canada vise le développement et la caractérisation de dérivés dipyrrométhène pour des applications dans le domaine du photovoltaïque. La quête du récoltage des photons se situant dans le proche-infrarouge a été au centre des modifications structurales explorées afin d’augmenter l’efficacité de conversion des cellules solaires de type organique et à pigments photosensibles. Trois familles de composés intégrant le motif dipyrrométhène ont été synthétisées et caractérisées du point de vue spectroscopique, électrochimique, structural ainsi que par modélisation moléculaire afin d’établir des relations structures-propriétés. La première famille comporte six azadipyrrométhènes au potentiel de coordination tétradentate sur des centres métalliques. Le développement d’une nouvelle voie synthétique asymétrique combinée à l’utilisation d’une voie symétrique classique ont permis d’obtenir l’ensemble des combinaisons de substituants possibles sur les aryles proximaux incluant les noyaux 2-hydroxyphényle, 2-méthoxyphényle et 2- pyridyle. La modulation du maximum d’absorption dans le rouge a pu être faite entre 598 et 619 nm. De même, la présence de groupements méthoxyle ou hydroxyle augmente l’absorption dans le violet (~410 nm) tel que démontré par modélisation. La caractérisation électrochimique a montré que les dérivés tétradentates étaient en général moins stables aux processus redox que leur contre-parti bidentate. La deuxième famille comporte dix dérivés BODIPY fusionnés de façon asymétrique en position [b]. L’aryle proximal a été modifié de façon systématique afin de mieux comprendre l’impact des substituents riches en électron et de la fusion de cycles aromatiques. De plus, ces dérivés ont été mis en relation avec une vaste série de composés analogues. Les résultats empiriques ont montré que les propriétés optoélectroniques de la plateforme sont régies par le degré de communication électronique entre l’aryle proximal, le pyrrole sur lequel il est attaché et le noyau indolique adjacent à ce dernier. Les maximums d’absorption dans le rouge sont modulables entre 547 et 628 nm et la fluorescence des composés se situe dans le proche- infrarouge. L’un des composé s’est révélé souhaitable pour une utilisation en photovoltaïque ainsi qu’à titre de sonde à pH. La troisième famille comporte cinq complexes neutres de RuII basés sur des polypyridines et portant un ligand azadipyrrométhène cyclométalé. Les composés ont montré une forte absorption de photons dans la région de 600 à 800 nm (rouge à proche- infrarouge) et qui a pu être étendue au-delà de 1100 nm dans le cas des dérivés portant un ligand terpyridine. L’analyse des propriétés optoélectroniques de façon empirique et théorique a montré un impact significatif de la cyclométalation et ouvert la voie pour leur étude en tant que photosensibilisateurs en OPV et en DSSC. La capacité d’un des complexes à photo-injecter un électron dans la bande de conduction du semi-conducteur TiO2 a été démontré en collaboration avec le groupe du Pr Gerald J. Meyer à University of North Carolina at Chapel Hill, premier pas vers une utilisation dans les cellules solaires à pigments photosensibles. La stabilité des complexes en solution s’est toutefois avérée problématique et des pistes de solutions sont suggérées basées sur les connaissances acquises dans le cadre de cette thèse.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Public perspective towards social impact of chang e lunar probe program

The present article is based on the MA thesis of Hou Bowen (Ph.D candidate) and on the presentation made at the ISA World Congress of Sociology held in Yokohama (Japan) on July 2014 at the Session on “Assessing Technologies: Global Patterns of Trust and Distrust” of RC23-Sociology of Science and Technology.

Twenty-four-hour esophageal pH monitoring in children and adolescents with chronic and/or recurrent rhinosinusitis

Gastroesophageal reflux (GER) disorder was studied in children and adolescents with chronic and/or recurrent rhinosinusitis not associated with bronchial asthma. Ten children with a clinical and radiological diagnosis of chronic and/or recurrent rhinosinusitis, consecutively attended at the Pediatric Otolaryngology Outpatient Clinic, Federal University of São Paulo, were evaluated. Prolonged esophageal pH monitoring was used to investigate GER disorder. The mean age of the ten patients evaluated (eight males) was 7.4 ± 2.4 years. Two patients presented vomiting as a clinical manifestation and one patient presented retrosternal pain with a burning sensation. Twenty-four-hour esophageal pH monitoring was performed using the Sandhill apparatus. An antimony probe electrode was placed in the lower third of the esophagus, confirmed by fluoroscopy and later by a chest X-ray. The parameters analyzed by esophageal pH monitoring included: total percent time of the presence of acid esophageal pH, i.e., pH below 4 (<4.2%); total number of acid episodes (<50 episodes); number of reflux episodes longer than 5 min (3 or less), and duration of the longest reflux episode (<9.2 min). One patient (1/10, 10%) presented a 24-h esophageal pH profile compatible with GER disorder. This data suggest that an association between chronic rhinosinusitis not associated with bronchial asthma and GER disorder may exist in children and adolescents, especially in those with compatible GER disorder symptoms. In these cases, 24-h esophageal pH monitoring should be performed before indicating surgery, since the present data suggest that 10% of chronic rhinosinusitis surgeries can be eliminated.

Cl- and regulation of pH by MDCK-C11 cells

The interaction between H+ extrusion via H+-ATPase and Cl- conductance was studied in the C11 clone of MDCK cells, akin to the intercalated cells of the collecting duct. Cell pH (pHi) was measured by fluorescence microscopy using the fluorescein-derived probe BCECF-AM. Control recovery rate measured after a 20 mM NH4Cl acid pulse was 0.136 ± 0.008 pH units/min (dpHi/dt) in Na+ Ringer and 0.032 ± 0.003 in the absence of Na+ (0 Na+). With 0 Na+ plus the Cl- channel inhibitor NPPB (10 µM), recovery was reduced to 0.014 ± 0.001 dpHi/dt. 8-Br-cAMP, known to activate CFTR Cl- channels, increased dpHi/dt in 0 Na+ to 0.061 ± 0.009 and also in the presence of 46 nM concanamycin and 50 µM Schering 28080. Since it is thought that the Cl- dependence of H+-ATPase might be due to its electrogenic nature and the establishment of a +PD (potential difference) across the cell membrane, the effect of 10 µM valinomycin at high (100 mM) K+ was tested in our cells. In Na+ Ringer, dpHi/dt was increased, but no effect was detected in 0 Na+ Ringer in the presence of NPPB, indicating that in intact C11 cells the effect of blocking Cl- channels on dpHi/dt was not due to an adverse electrical gradient. The effect of 100 µM ATP was studied in 0 Na+ Ringer solution; this treatment caused a significant inhibition of dpHi/dt, reversed by 50 µM Bapta. We have shown that H+-ATPase present in MDCK C11 cells depends on Cl- ions and their channels, being regulated by cAMP and ATP, but not by the electrical gradient established by electrogenic H+ transport.

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.

Mikrostrukturierte Sensoren für die pH-Wert-Bestimung auf Basis von dünnen, intrinisch leitfähigen Polyanilinschichten

Der pH-Wert stellt in der Chemie, Physik, Biologie, Pharmazie und Medizin eine wichtige Meßgröße dar, da eine Vielzahl von Reaktionen durch den pH-Wert bestimmt wird. In der Regel werden zur pH-Wert-Messung Glaselektroden eingesetzt. Hierbei konnte der pH-sensitive Bereich zwar bis auf einige Mikrometer reduziert werden, aber die Gesamtab-messungen betragen immer noch 15-20 cm. Mit der Einführung miniaturisierter Reaktionsgefäße ist daher der Bedarf an miniaturisierten Sensoren enorm gestiegen. Um in solchen Gefäßen Reaktionsparameter wie z. B. den pH-Wert zu kontrollieren, müssen die Gesamtabmessungen der Sensoren verringert werden. Dies lässt sich mit Hilfe der Mikrostrukturtechnik von Silizium realisieren. Hiermit lassen sich Strukturen und ganze Systeme bis in den Nanometerbereich herstellen. Basierend auf Silizium und Gold als Elektrodenmaterial wurden im Rahmen dieser Arbeit verschiedene Interdigitalstrukturen hergestellt. Um diese Strukturen zur pH-Wert-Messungen einsetzen zu können, müssen sie mit einer pH-sensitiven Schicht versehen werden. Hierbei wurde Polyanilin, ein intrinsisch leitendes Polymer, aufgrund seine pH-abhängigen elektrischen und optischen Verhaltens eingesetzt. Die Beschichtung dieser Sensoren mit Polyanilin erfolgte vorwiegend elektrochemisch mit Hilfe der Zyklovoltammetrie. Neben der Herstellung reiner Polyanilinfilme wurden auch Kopolymerisationen von Anilin und seinen entsprechenden Aminobenzoesäure- bzw. Aminobenzensulfonsäurederivaten durchgeführt. Ergebnisse dazu werden vorgestellt und diskutiert. Zur Charakterisierung der resultierenden Polyanilin- und Kopolymerfilme auf den Inter-digitalstrukturen wurden mit Hilfe der ATR-FT-IR-Spektroskopie Spektren aufgenommen, die gezeigt und diskutiert werden. Eine elektrochemische Charakterisierung der Polymere erfolgte mittels der Zyklovoltammetrie. Die mit Polyanilin bzw. seinen Kopolymeren beschichteten Sensoren wurden dann für Widerstandsmessungen an den Polymerfilmen in wässrigen Medien eingesetzt. Polyanilin zeigt lediglich eine pH-Sensitivität in einem pH-Bereich von pH 2 bis pH 4. Durch den Einsatz der Kopolymere konnte dieser pH-sensitive Bereich jedoch bis zu einem pH-Wert von 10 ausgeweitet werden. Zur weiteren Miniaturisierung der Sensoren wurde das Konzept der interdigitalen Elektroden-paare auf Cantilever übertragen. Die pH-sensitive Zone konnte dabei auf 500 µm2 bei einer Gesamtlänge des Sensors (Halter mit integriertem Cantilever) von 4 mm reduziert werden. Neben den elektrischen pH-abhängigen Eigenschaften können auch die optischen Eigen-schaften des Polyanilins zur pH-Detektion herangezogen werden. Diese wurden zunächst mit Hilfe der UV-VIS-Spektroskopie untersucht. Die erhaltenen Spektren werden gezeigt und kurz diskutiert. Mit Hilfe eines Raster-Sonden-Mikroskops (a-SNOM, Firma WITec) wurden Reflexionsmessungen an Polyanilinschichten durchgeführt. Zur weiteren Miniaturisierung wurden Siliziumdioxidhohlpyramiden (Basisfläche 400 µm2) mit Spitzenöffnungen in einem Bereich von 50-150 nm mit Polyanilin beschichtet. Auch hier sollten die optischen Eigenschaften des Polyanilins zur pH-Wert-Sensorik ausgenutzt werden. Es werden erste Messungen an diesen Strukturen in Transmission diskutiert.

Self-assembly of a modified amyloid peptide fragment: pH-responsiveness and nematic phase formation

The self-assembly of peptide YYKLVFFC based on a fragment of the amyloid beta (A) peptide, A beta 16-20, KLVFF has been studied in aqueous solution. The peptide is designed with multiple functional residues to examine the interplay between aromatic interactions and charge on the self-assembly, as well as specific transformations such as the pH-induced phenol-phenolate transition of the tyrosine residue. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopies are used to investigate the conditions for beta-sheet self-assembly and the role of aromatic interactions in the CD spectrum as a function of pH and concentration. The formation of well-defined fibrils at pH 4.7 is confirmed by cryo-TEM (transmission electron microscope) and negative stain TEM. The morphology changes at higher pH, and aggregates of short twisted fibrils are observed at pH 11. Polarized optical microscopy shows birefringence at a low concentration (1 wt.-%) of YYKLVFFC in aqueous solution, and small-angle X-ray scattering was used to probe nematic phase formation in more detail. A pH-induced transition from nematic to isotropic phases is observed on increasing pH that appears to be correlated to a reduction in aggregate anisotropy upon increasing pH.

The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells

The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (J(H)) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 mu M, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of J(H) by the NHE3 inhibitor S3226 (1 mu M) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 mu M EIPA abolished J(H) entirely, but NHE4 was not inhibited at 10 mu M; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of J(H). Cl--dependent NHE was not detected: in 0 Cl- solutions J(H) was increased and not reduced. In 0 Cl- cell volume decreased significantly, which was abolished by the Cl- channel blocker NPPB, indicating that the 0 Cl- effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

Laryngeal and voice disorders in patients with gastroesophageal symptoms. Correlation with pH-monitoring

OBJETIVO: Investigar as alterações laríngeas e vocais em pacientes com sintomas de refluxo gastroesofágico e correlacioná-las com o exame de phmetria. MÉTODOS: Estudo prospectivo que incluiu os pacientes atendidos nos ambulatórios de Distúrbios da Voz da Faculdade de Medicina de Botucatu no período de cinco anos com sintomas vocais e gastroesofágicos. Os pacientes foram submetidos à videolaringoscopia, às análises vocais perceptivo-auditivas, a analise vocal acústica computadorizada e ao exame de pHmetria de dois canais com monitorização durante 24 horas. RESULTADOS: Foram incluídos 57 pacientes (entre 21 a 65 anos; 45 mulheres e 12 homens). Desses, 18 apresentavam pHmetria normal (31,6%) e 39 alterada (68,4%). As videolaringoscopias registraram diversas lesões laríngeas tanto nos pacientes com pHmetria normal como alterada, sendo mais relevantes neste último grupo, destacando-se a paquidermia posterior. As avaliações vocais perceptivo-auditivas identificaram alterações vocais de diversas intensidades em ambos os grupos, mais importantes nos pacientes com pHmetria alterada. Todos os parâmetros acústicos, exceto Fo, mostraram-se alterados em ambos os grupos, quando comparados aos controles. CONCLUSÕES: Alterações vocais perceptivas e acústicas, e lesões laríngeas foram registradas tanto nos pacientes com phmetria normal como alterada, sinalizando para a importância da historia clínica e dos achados videolaringoscópicos no diagnóstico das laringites ácidas.

pH at the micellar interface: Synthesis of pH probes derived from salicylic acid, acid-base dissociation in sodium dodecyl sulfate micelles, and Poisson-Boltzmann simulation

The study of the H+ concentration at the micellar interface is a convenient system for modeling the distribution of H+ at interfaces. We have synthesized salicylic acid derivatives to analyze the proton dissociation of both the carboxylic and phenol groups of' the probes, determining spectrophotometrically the apparent pK(a)'s (pK(ap)) in sodium dodecyl Sulfate, SDS, micelles with and without added salt. The synthesized probes were 2-hydroxy-5-(2-trimethylammoniumacetyl)benzoate; 2-hydroxy-5-(2-dimethylhexadecylammoniumacetyl)benzoate- 2-hydroxy-5-(2-dimethylhexadecylammoniumhexanoyl)benzoate-, 2-hydroxy-5-(2-diniethylhexadecylammoniumundecanoyl)betizoate; 2-hydroxy-5-acetylbenzoic acids and 2-hydroxy-5-dodecanoylbenzoic acid. Upon incorporation into SDS micelles the pK(ap)'s of both carboxylic and phenol groups increased by ca. 3 pH units and NaCl addition caused a decrease in the probe-incorporated pKap. The experimental results were fitted with a cell model Poisson-Boltzmann (P-B) equation taking in consideration the effect of salt on the aggregation number of SDS and using the distance of' the dissociating group as a parameter. The conformations of the probes were analyzed theoretically using two dielectric constants, e.g., 2 and 78. Both the P-B analysis and conformation calculations can be interpreted by assuming that the acid groups dissociate very close to, or at, the interface. Our results are consistent with the assumption that the intrinsic pK(a)'s of both carboxylic and phenol groups of the salicylic acid probes used here can be taken as those in water. Using this assumption the micellar and salt effects on the pKap's of the (trialkylammonium)benzoate probes were described accurately using a cell model P-B analysis. (c) 2005 Elsevier B.V. All rights reserved.