Final results from the PERUSE study of first-line pertuzumab plus trastuzumab plus a taxane for HER2-positive locally recurrent or metastatic breast cancer, with a multivariable approach to guide prognostication.

BACKGROUND: The phase III CLinical Evaluation Of Pertuzumab And TRAstuzumab (CLEOPATRA) trial established the combination of pertuzumab, trastuzumab and docetaxel as standard first-line therapy for human epidermal growth factor receptor 2 (HER2)-positive locally recurrent/metastatic breast cancer (LR/mBC). The multicentre single-arm PERtUzumab global SafEty (PERUSE) study assessed the safety and efficacy of pertuzumab and trastuzumab combined with investigator-selected taxane in this setting. PATIENTS AND METHODS: Eligible patients with inoperable HER2-positive LR/mBC and no prior systemic therapy for LR/mBC (except endocrine therapy) received docetaxel, paclitaxel or nab-paclitaxel with trastuzumab and pertuzumab until disease progression or unacceptable toxicity. The primary endpoint was safety. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). Prespecified subgroup analyses included subgroups according to taxane, hormone receptor (HR) status and prior trastuzumab. Exploratory univariable analyses identified potential prognostic factors; those that remained significant in multivariable analysis were used to analyse PFS and OS in subgroups with all, some or none of these factors. RESULTS: Of 1436 treated patients, 588 (41%) initially received paclitaxel and 918 (64%) had HR-positive disease. The most common grade ≥3 adverse events were neutropenia (10%, mainly with docetaxel) and diarrhoea (8%). At the final analysis (median follow-up: 5.7 years), median PFS was 20.7 [95% confidence interval (CI) 18.9-23.1] months overall and was similar irrespective of HR status or taxane. Median OS was 65.3 (95% CI 60.9-70.9) months overall. OS was similar regardless of taxane backbone but was more favourable in patients with HR-positive than HR-negative LR/mBC. In exploratory analyses, trastuzumab-pretreated patients with visceral disease had the shortest median PFS (13.1 months) and OS (46.3 months). CONCLUSIONS: Mature results from PERUSE show a safety and efficacy profile consistent with results from CLEOPATRA and median OS exceeding 5 years. Results suggest that paclitaxel is a valid alternative to docetaxel as backbone chemotherapy. Exploratory analyses suggest risk factors that could guide future trial design.

Preliminary safety and efficacy of first-line pertuzumab combined with trastuzumab and taxane therapy for HER2-positive locally recurrent or metastatic breast cancer (PERUSE).

BACKGROUND: Pertuzumab combined with trastuzumab and docetaxel is the standard first-line therapy for HER2-positive metastatic breast cancer, based on results from the phase III CLEOPATRA trial. PERUSE was designed to assess the safety and efficacy of investigator-selected taxane with pertuzumab and trastuzumab in this setting. PATIENTS AND METHODS: In the ongoing multicentre single-arm phase IIIb PERUSE study, patients with inoperable HER2-positive advanced breast cancer (locally recurrent/metastatic) (LR/MBC) and no prior systemic therapy for LR/MBC (except endocrine therapy) received docetaxel, paclitaxel or nab-paclitaxel with trastuzumab [8 mg/kg loading dose, then 6 mg/kg every 3 weeks (q3w)] and pertuzumab (840 mg loading dose, then 420 mg q3w) until disease progression or unacceptable toxicity. The primary end point was safety. Secondary end points included overall response rate (ORR) and progression-free survival (PFS). RESULTS: Overall, 1436 patients received at least one treatment dose (initially docetaxel in 775 patients, paclitaxel in 589, nab-paclitaxel in 65; 7 discontinued before starting taxane). Median age was 54 years; 29% had received prior trastuzumab. Median treatment duration was 16 months for pertuzumab and trastuzumab and 4 months for taxane. Compared with docetaxel-containing therapy, paclitaxel-containing therapy was associated with more neuropathy (all-grade peripheral neuropathy 31% versus 16%) but less febrile neutropenia (1% versus 11%) and mucositis (14% versus 25%). At this preliminary analysis (52 months' median follow-up), median PFS was 20.6 [95% confidence interval (CI) 18.9-22.7] months overall (19.6, 23.0 and 18.1 months with docetaxel, paclitaxel and nab-paclitaxel, respectively). ORR was 80% (95% CI 78%-82%) overall (docetaxel 79%, paclitaxel 83%, nab-paclitaxel 77%). CONCLUSIONS: Preliminary findings from PERUSE suggest that the safety and efficacy of first-line pertuzumab, trastuzumab and taxane for HER2-positive LR/MBC are consistent with results from CLEOPATRA. Paclitaxel appears to be a valid alternative taxane backbone to docetaxel, offering similar PFS and ORR with a predictable safety profile. CLINICALTRIALS.GOV: NCT01572038.

Rabies in North Africa and the Middle East: current situation, strategies and outlook. / La rage dans les pays d'Afrique du Nord et du Moyen-Orient : situation actuelle et perspectives.

In the Middle East and North Africa (MENA) region, dogs are the main reservoir for rabies. In this region, rabies affects more domestic carnivores (50% of cases) than farm animals (40% of cases). Rabies in large livestock animals, which are infected mainly by dogs, results in economic losses, undermines food safety and poses a risk for humans. In the MENA region, rabies is largely an urban problem, the virus being transmitted to humans by free-roaming dogs, 30% of which are less than one year old. In cities, the density of the free-roaming dog population varies between 0.6 and 1.5 dogs per km2, and almost double this figure in suburban and rural areas, where 80% of canine rabies cases occur. In the Maghreb, the annual average number of confirmed human deaths from rabies is around 47, showing a strong correlation with the number of animal cases declared (approximately 1,442 cases a year). In the Middle East, only a few rabies cases in humans are officially reported, suggesting that rabies cases in humans are grossly underreported. National strategies to control rabies include vaccination, controlling the freeroaming dog population and raising awareness among the human population at risk. Owing to limited resources and poor coordination between the different stakeholders, these strategies are only partially implemented. The Food and Agriculture Organization of the United Nations, in close collaboration with the World Organisation for Animal Health and the World Health Organization, supports countries in formulating and implementing coordinated strategies between the ministries concerned, local authorities and non-governmental organisations.

Dans la région Moyen-Orient et Afrique du Nord, le chien est le principal réservoir de la rage. En effet, dans cette région, la rage affecte plus de carnivores domestiques (50 % des cas) que d'animaux de ferme (40 % des cas). L'infection des animaux d'élevage est essentiellement transmise par les chiens et entraîne des pertes économiques, un impact négatif sur la sécurité alimentaire et un risque pour les humains. Dans la région Moyen-Orient et Afrique du Nord, la rage est un problème principalement urbain, le virus étant transmis aux humains par des chiens errants dont 30 % sont âgés de moins d'un an. La densité de la population de chiens errants varie de 0,6 à 1,5 chien par km² dans les villes et à peu près le double dans les zones périurbaines et rurales où sont enregistrés 80 % des cas de rage canine. Au Maghreb, le nombre annuel moyen de cas confirmés de décès humains par la rage est d'environ 47, avec une forte corrélation avec le nombre de cas chez les animaux qui avoisine 1 442 cas confirmés par an. Au Moyen-Orient, seuls quelques cas de rage chez l'homme sont officiellement déclarés ce qui suggère que les cas de rage chez les humains sont largement sous-déclarés. Les stratégies nationales de lutte contre la rage comprennent la vaccination, le contrôle des populations de chiens errants et la sensibilisation des populations humaines au risque d'infection. Ces stratégies sont partiellement mises en oeuvre en raison des ressources limitées et du manque de coordination entre les différents intervenants. L'Organisation des Nations Unies pour l'alimentation et l'agriculture (FAO), en étroite collaboration avec l'Organisation mondiale de la santé animale (OIE) et l'Organisation mondiale de la santé (OMS), soutient les pays dans l'élaboration et la mise en oeuvre de stratégies coordonnées entre les ministères concernés, les autorités locales et les organisations non gouvernementales.

En la región del Oriente Medio y África del Norte, el perro es el principal reservorio de la rabia. En estas zonas, en efecto, la rabia afecta a un mayor número de carnívoros domésticos (un 50% de los casos) que de animales de granja (un 40% de los casos). El perro es la vía fundamental por la que los animales de producción pecuaria contraen la infección, que provoca pérdidas económicas, hace mella en la seguridad alimentaria y entraña un riesgo sanitario para todas las personas. En esta región la rabia es un problema esencialmente urbano, por cuanto la vía de transmisión del virus a las personas son perros vagabundos, de los que el 30% tiene menos de un año de edad. La densidad de población de estos perros varía: de 0,6 a 1,5 individuos por km² en las ciudades hasta aproximadamente el doble en las zonas periurbanas y rurales, donde se producen el 80 % de los casos de rabia canina. En el Magreb se registra un promedio anual de unos 47 casos confirmados de muerte humana por rabia, cifra que presenta una estrecha correlación con el número de casos confirmados que se dan anualmente en los animales, próximo a los 1.442 casos. En el Oriente Medio se declaran oficialmente contados casos humanos de rabia, lo que lleva a pensar que el número real de casos en el ser humano supera con creces el de casos declarados. Las estrategias nacionales de lucha contra la rabia prevén medidas de vacunación, control de las poblaciones de perros vagabundos y sensibilización de la población humana expuesta al riesgo de infección. Esas estrategias se aplican solo parcialmente debido a la escasez de recursos y a la falta de coordinación entre las distintas instancias encargadas de ello. La Organización de las Naciones Unidas para la Alimentación y la Agricultura (FAO), en estrecha colaboración con la Organización Mundial de Sanidad Animal (OIE) y la Organización Mundial de la Salud (OMS), respalda a los países en la elaboración y aplicación de estrategias coordinadas entre los ministerios competentes, las autoridades locales y las organizaciones no gubernamentales.

Review of rabies situation and control in the North African region with a focus on Tunisia.

Rabies is a major zoonosis that affects the central nervous system of warm-blooded mammals. The disease is present worldwide, except for some islands. Africa and Asia record over 95% of the fatal cases of rabies worldwide, and therefore the greatest risk to human life from rabies occurs in these regions. Mass vaccination of dogs is the most appropriate way to control and eliminate the disease at the animal source, in order to interrupt the infectious cycle of the disease from animals to humans. Rabies is endemic in the North African region, and countries should be encouraged to develop programmes for eliminating human rabies through the implementation of sustained campaigns to immunise dogs and by providing post-exposure prophylaxis (PEP) to people who have been exposed to suspected rabid dogs. In Tunisia, the national strategy against rabies was started in 1981 and it has been upgraded since. Following the launch of the annual vaccination programme in 1993, there was a significant improvement in the health status for rabies in Tunisia, with a decrease in the number of cases in animals and humans. Since 2011, an increase in cases of rabies in dogs and humans has been observed, due to lower vaccination coverage, mismanagement of waste and an increase in the stray dog population. The political will at international, regional and national levels is the cornerstone of the strategy to eliminate the disease. In the framework of the regional approach in the Maghreb, additional efforts and political willingness are necessary at the national level to better control and eventually eliminate rabies.

La rage est une zoonose majeure affectant le système nerveux central des mammifères à sang chaud. À l'exception de quelques territoires insulaires, la maladie est présente dans le monde entier. L'Afrique et l'Asie enregistrant plus de 95 % des décès dus à la rage dans le monde, c'est dans ces deux régions que la rage représente le plus grand risque pour l'être humain. La vaccination massive des chiens est la méthode indiquée pour lutter contre cette maladie et l'éliminer à sa source animale, ce qui permet de mettre un terme au cycle de transmission des animaux à l'homme. En Afrique du Nord, la rage est endémique et il faut encourager les pays de la région à introduire des programmes d'élimination de la rage humaine basés sur des campagnes soutenues d'immunisation des chiens et sur l'administration d'une prophylaxie post-exposition aux personnes exposées à des chiens suspectés de rage. La Tunisie a mis en place en 1981 une stratégie nationale de lutte contre la rage et l'a actualisée par la suite. Suite au lancement en 1993 d'un programme de vaccination annuelle, la situation sanitaire de la Tunisie à l'égard de la rage s'est améliorée et le nombre de cas enregistrés chez l'homme et chez les animaux a fortement décliné. Toutefois, depuis 2011 ce nombre est reparti à la hausse chez le chien et chez l'homme en raison d'une couverture vaccinale insatisfaisante, d'une gestion calamiteuse des déchets et de l'augmentation concomitante de la population de chiens errants. La volonté politique au niveau international, régional et national est la pierre angulaire de la stratégie d'élimination de cette maladie. Dans le cadre de l'approche régionale adoptée par le Maghreb, des efforts accrus et une véritable volonté politique sont nécessaires à l'échelle nationale pour mieux contrôler la rage et, à terme, pour l'éliminer.

La rabia es una importante zoonosis que afecta al sistema nervioso central de los mamíferos, animales de sangre caliente. Con la salvedad de algunas islas, está presente en el mundo entero. En África y Asia se registran más del 95% de los casos mortales de rabia que tienen lugar en el mundo, por lo que el máximo nivel de riesgo para la vida humana derivado de esta enfermedad se da en dichas regiones. La vacunación masiva de perros es el medio más adecuado para controlar y eliminar la rabia en su fuente animal, pues con ello se interrumpe el ciclo infeccioso de la enfermedad que va de los animales al hombre. La rabia es endémica en el Norte de África, por lo que es preciso alentar a los países de esta región a que elaboren programas destinados a eliminar la rabia humana mediante la realización de campañas duraderas para inmunizar a la población canina y la aplicación de medidas de profilaxis tras la exposición a toda persona expuesta a perros presuntamente rabiosos. Desde que fue puesta en marcha en 1981, la estrategia nacional de Túnez contra la rabia ha sido regularmente actualizada. A raíz de la implantación en 1993 del programa anual de vacunaciones, la situación sanitaria del país con respecto a la enfermedad mejoró sustancialmente, lo que se plasmó en una caída del número de casos en personas y animales. Desde 2011 se viene observando un aumento del número de casos de rabia en perros y humanos, hecho que se explica por una menor cobertura de vacunación, una gestión incorrecta de los desechos y un crecimiento de la población de perros vagabundos. La voluntad política a escala internacional, regional y nacional es la piedra angular de toda estrategia para eliminar la enfermedad. Para luchar más eficazmente contra la rabia y a la postre conseguir eliminarla los países, a la par que inscriben su labor en los planteamientos regionales aplicados en el Magreb, deben redoblar esfuerzos y hacer efectiva una mayor voluntad política.

Valvular dystrophy associated filamin A mutations reveal a new role of its first repeats in small-GTPase regulation.

Filamin A (FlnA) is a ubiquitous actin binding protein which anchors various transmembrane proteins to the cell cytoskeleton and provides a scaffold to many cytoplasmic signaling proteins involved in actin cytoskeleton remodeling in response to mechanical stress and cytokines stimulation. Although the vast majority of FlnA binding partners interact with the carboxy-terminal immunoglobulin like (Igl) repeats of FlnA, little is known on the role of the amino-N-terminal repeats. Here, using cardiac mitral valvular dystrophy associated FlnA-G288R and P637Q mutations located in the N-terminal Igl repeat 1 and 4 respectively as a model, we identified a new role of FlnA N-terminal repeats in small Rho-GTPases regulation. Using FlnA-deficient melanoma and HT1080 cell lines as expression systems we showed that FlnA mutations reduce cell spreading and migration capacities. Furthermore, we defined a signaling network in which FlnA mutations alter the balance between RhoA and Rac1 GTPases activities in favor of RhoA and provided evidences for a role of the Rac1 specific GTPase activating protein FilGAP in this process. Together our work ascribed a new role to the N-terminal repeats of FlnA in Small GTPases regulation and supports a conceptual framework for the role of FlnA mutations in cardiac valve diseases centered around signaling molecules regulating cellular actin cytoskeleton in response to mechanical stress.

Expression of the familial cardiac valvular dystrophy gene, filamin-A, during heart morphogenesis.

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.

Electronic temperatures, densities, and plasma x-ray emission of a 14.5 GHz electron-cyclotron resonance ion source.

We have performed a systematic study of the bremsstrahlung emission from the electrons in the plasma of a commercial 14.5 GHz electron-cyclotron resonance ion source. The electronic spectral temperature and the product of ionic and electronic densities of the plasma are measured by analyzing the bremsstrahlung spectra recorded for several rare gases (Ar, Kr, and Xe) as a function of the injected power. Within our uncertainty, we find an average temperature of approximately 48 keV above 100 W, with a weak dependency on the injected power and gas composition. Charge state distributions of extracted ion beams have been determined as well, providing a way to disentangle the ionic density from the electronic density. Moreover x-ray emission from highly charged argon ions in the plasma has been observed with a high-resolution mosaic-crystal spectrometer, demonstrating the feasibility for high-precision measurements of transition energies of highly charged ions, in particular, of the magnetic dipole (M1) transition of He-like of argon ions.

Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.

Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosine-based basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the mu subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in mu subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4-mu 3A interaction and enhancing AQP4-lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression.

[Bisprolol in heart failure: efficacy and costs in a French setting according to CIBIS II]. / Bisoprolol dans l'insuffisance cardiaque: efficacité et coûts dans le cadre français selon CIBIS II.

After a significant mortality benefit with bisoprolol in heart failure was demonstrated in CIBIS-II, an economic evaluation has been performed in cost-effectiveness terms. Average direct costs per patient were based on clinical data from 231 French patients, and measured in the perspective of the French National Health Insurance, effectiveness being expressed in terms of life days gained per patient. The extra cost of bisoprolol treatment and follow-up (averaging FF 1300 per 1.3 years) is outweighed by the reduction in hospitalization costs (representing a saving of FF 10,500 per patient) and other medication costs. Finally, bisoprolol therapy induces benefits in terms of both cost and survival: on average FF 9500 and 11 life days per patient. Sensitivity analyses confirm these results.

Basolateral membrane expression of a K+ channel, Kir 2.3, is directed by a cytoplasmic COOH-terminal domain.

The inwardly rectifying potassium channel Kir 2.3 is specifically targeted and expressed on the basolateral membrane of certain renal epithelial cells. In the present study, the structural basis for polarized targeting was elucidated. Deletion of a unique COOH-terminal domain produced channels that were mistargeted to the apical membrane, consistent with the removal of a basolateral membrane-sorting signal. By characterizing a series of progressively smaller truncation mutants, an essential targeting signal was defined (residues 431-442) within a domain that juxtaposes or overlaps with a type I PDZ binding motif (442). Fusion of the COOH-terminal structure onto CD4 was sufficient to change a random membrane-trafficking and expression pattern into a basolateral membrane one. Using metabolic labeling and pulse-chase and surface immunoprecipitation, we found that CD4-Kir2.3 COOH-terminal chimeras were rapidly and directly targeted to the basolateral membrane, consistent with a sorting signal that is processed in the biosynthetic pathway. Collectively, the data indicate that the basolateral sorting determinant in Kir 2.3 is composed of a unique arrangement of trafficking motifs, containing tandem, conceivably overlapping, biosynthetic targeting and PDZ-based signals. The previously unrecognized domain corresponds to a highly degenerate structure within the Kir channel family, raising the possibility that the extreme COOH terminus of Kir channels may differentially coordinate membrane targeting of different channel isoforms.

Electropharmacological characterization of cardiac repolarization in German shepherd dogs with an inherited syndrome of sudden death: abnormal response to potassium channel blockers.

OBJECTIVES: This study sought to determine whether abnormal ventricular repolarization is implicated in cardiac arrhythmias of German shepherd dogs with inherited sudden death. BACKGROUND: Moïse et al. (9) have identified German shepherd dogs that display pause-dependent lethal ventricular arrhythmias. METHODS: Ventricular repolarization was studied both in vivo using electrocardiogram recordings on conscious dogs and in vitro with a standard microelectrode technique performed on endomyocardial biopsies and Purkinje fibers. Pharmacological manipulation was used to evaluate the role of potassium channels. RESULTS: In control conditions, electrocardiogram parameters were similar in both groups of dogs, except for the PR interval (18% longer in affected dogs, p < 0.05). Injection of d,l-sotalol (2 mg/kg) prolonged QT interval more in affected dogs (+14%, n = 9) than it did in unaffected dogs (+6%, n = 6, p < 0.05) and increased the severity of arrhythmias in affected dogs. In vitro, in control conditions, action potential duration (APD90) of endomyocardial biopsies and Purkinje fibers were significantly longer in affected dogs (respectively 209 +/- 3 ms, n = 30 and 352 +/- 15 ms, n = 17) than they were in unaffected dogs (197 +/- 4 ms, n = 25 and 300 +/- 9 ms, n = 30) at a pacing cycle length (PCL) of 1,000 ms. This difference increased with PCL. The kinetics of adaptation of APD90 to a change in PCL was faster in affected dogs. D,l-sotalol (10(-5) and 10(-4)M) increased APD90 in both groups of dogs, but this increase was greater in affected dogs, with the occurrence of triggered activity on Purkinje fibers. E-4031 (10(-7) and 10(-6) M), an I(Kr)-blocker, increased APD90 similarly in both groups of dogs. Chromanol 293B (10(-6) and 10(-5)M), an I(Ks)-blocker, increased significantly APD90 in unaffected dogs but had no effect in affected dogs. CONCLUSIONS: These results support the hypothesis of an abnormal cardiac repolarization in affected dogs. The effects of 293B suggest that I(Ks) may be involved in this anomaly.

Water permeability in rat oocytes at different maturity stages: aquaporin-9 expression.

Important functional and structural modifications occur in mammalian oocytes during their arrival to maturity. In this process, oocytes switch from a high activity level, implying an important metabolic rate and a coordinated movement of water and solutes, to a lower functional state. The aim of this work was to study the mechanisms involved in water movements during oocyte arrival to maturity. Volume changes, induced by an osmotic gradient, were followed by video microscopy in rat oocytes. The water osmotic permeability (P(osm)) of immature oocytes (proestrus) was sensitive to HgCl(2) and phloretin. In contrast, mature oocytes (estrus) had a reduced P(osm) that was not sensitive to these compounds. When proestrus oocytes were incubated in vitro at 37 degrees C they spontaneously arrived at maturity and its P(osm) decreased between four and six hours of incubation. RT-PCR experiments were performed using specific primers for all rat aquaporins that had been cloned. We found that aquaporin-9 transcript (AQP9) is present in proestrus oocytes but not in estrus oocytes. AQP9 has been recently described as a "broad selective channel" responsible for solute and water transfers in highly active cells. Our experiments showed that proestrus oocytes, but not estrus, are permeable to mannitol. It is concluded that during the process of maturation, P(osm) decreases and AQP9 transcripts disappear. We report here the first study correlating water permeability and aquaporin mRNA expression in mammalian oocytes.

A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) channels in a closed state.

Mutations in the inward rectifying renal K(+) channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332-391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP-wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332-351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.

Abnormal cardiac repolarization and impulse initiation in German shepherd dogs with inherited ventricular arrhythmias and sudden death.

OBJECTIVE: We tested the hypothesis that delayed afterdepolarization (DAD)-associated rhythms in German shepherd dogs with reduced anteroseptal left ventricular (LV) sympathetic innervation derive from abnormal beta-adrenergic receptor effector coupling. METHODS AND RESULTS: In anteroseptal LV midmyocardium of afflicted dogs, beta-receptor density was greater than that in normal dogs (P < .05), with affinity being equal in both groups. Basal and maximum isoproterenol (ISO) stimulated adenylyl cyclase activity of anteroseptal LV of afflicted dogs was greater than that in normal dogs (P < .05). Isolated anteroseptal M cell preparations of afflicted dogs studied with microelectrodes showed abnormal lengthening, rather than shortening of action potential duration in response to ISO, as well as a 61% incidence of 10(-7) mol/l ISO-induced triggered activity as compared to 12% in normals (P < .05). In contrast, there was no difference between afflicted and control dogs in triggered activity, beta-receptors or adenylyl cyclase activity in a normally innervated region of the ventricles. CONCLUSION: In this model there is an increase in beta-receptor density and beta-adrenergic stimulation of adenylyl cyclase and of triggered activity in anteroseptal myocardium but not in a normally innervated region of the heart. Hence, abnormal beta-adrenergic signal transduction appears associated with the neural abnormality identified in dogs with inherited VT.

Processing and transport of ROMK1 channel is temperature-sensitive.

To investigate the biosynthetic mechanisms involved in the expression of the renal epithelial inward rectifying K(+) channel, ROMK1 (Kir1.1a), a six amino acid epitope (AU1) was introduced onto the extreme N-terminus for efficient immunoprecipitation. As expressed in Xenopus oocytes, the AU1 epitope did not modify the functional properties of the ROMK1 channel. To analyze kinetics of ROMK1 synthesis in renal epithelial cells, the AU1-ROMK1 construct was stably transfected in MDCK cells and pulse chase experiments were conducted. When the cells are grown at 37 degrees C, the ROMK1 protein was unstable, being rapidly degraded with a t(1/2) < 1 hour. Furthermore, whole cell patch clamp experiments failed to detect functional ROMK1 channels at the plasma membrane in cells grown at 37 degrees C. In contrast, the degradation process was minimized when the cells were grown at 26 degrees C (t(1/2) > 4 hours), allowing ROMK1 channels to be functionally expressed on the plasma membrane. In summary, in a mammalian epithelial expression system maintained at a physiological temperature, wild-type ROMK1 is bio-synthetically labile and incapable of efficient traffic to the plasmalemma. These observations are reminiscent of temperature sensitive biosynthetic defects in mutant plasma membrane proteins, suggesting that wild-type ROMK1 may require other factors, like the association of a surrogate subunit, for appropriate biosynthetic processing.

Effects of chronic treatment by amiodarone on transmural heterogeneity of canine ventricular repolarization in vivo: interactions with acute sotalol.

OBJECTIVE: The present study was designed to examine the effects of chronic amiodarone on the different ventricular cell subtypes in situ and to evaluate its interactions with sotalol. METHODS: Three groups of dogs were studied. Group I (n = 8) received no treatment. Group II (n = 7) and group III (n = 8) received, respectively, 100 and 200 mg amiodarone orally twice a day for 6 weeks to 8 months. In vivo studies were performed under halothane anesthesia 14 h after the last administration of amiodarone. Three leads ECG, femoral blood pressure and left ventricular intramural monophasic action potentials (MAP) were continuously recorded. Bradycardia was obtained by clamping the sinus node and beta-blockade and the heart was driven by atrial pacing. Three weeks before the in vivo experiments, the cellular electrophysiologic properties of right ventricular tissues obtained by cardiac biopsy in six treated and six control dogs were studied with standard microelectrodes. RESULTS: Amiodarone produced a dose-dependent decrease in plasma levels of triiodothyronine (T3; 5.9 +/- 0.4 pM in control dogs, 3.1 +/- 0.2 pM in group III, P < 0.001) without affecting thyroxine (T4). Under anesthesia, the QT interval was 14% larger in group III compared to group I at a paced cycle length (PCL) of 1500 ms (P < 0.05). This is consistent with the 10% increase in endocardial MAP duration in group III at the same PCL (P < 0.05). There was no significant increase in transmural dispersion of MAP duration. In group I, sotalol induced a significant reverse use-dependent increase in MAP duration. This effect was reduced in group II and completely suppressed in group III. Amiodarone prevented the sotalol-induced increase in transmural dispersion of ventricular repolarization which was 69 +/- 12 ms in untreated dogs, 41 +/- 8 ms in group II (P < 0.05) and 34 +/- 8 ms (P < 0.05) in group III at PCL = 1500 ms. Amiodarone also prevented the sotalol-induced ventricular tachyarrhythmias. In vitro, the action potential duration was longer in amiodarone-treated dogs that in control ones (208 +/- 5 ms versus 188 +/- 9 ms at PCL = 1000 ms, P < 0.05). The sotalol-induced prolongation of repolarization was reduced in amiodarone-treated dogs. CONCLUSION: Chronic treatment of dogs with amiodarone induced a moderate prolongation of the QT interval and MAP duration without affecting transmural dispersion of repolarization and inhibited the effects of acute sotalol, including the prolongation of repolarization, the increase in transmural dispersion of repolarization and the induction of arrhythmias.

Quality of life in multiple sclerosis in France, Germany, and the United Kingdom. Cost of Multiple Sclerosis Study Group.

OBJECTIVE: To assess the quality of life (QoL) of patients with multiple sclerosis in France, Germany, and the United Kingdom with a cross sectional study. METHODS: Patients were classified into three severity groups according to the expanded disability severity scale (EDSS); stage I, II, and III, corresponding to mild (EDSS 1.0-3.5), moderate (EDSS 4.0-6.0), or severe (EDSS 6.5-8.0) multiple sclerosis respectively. Ninety patients with multiple sclerosis and 30 control patients without multiple sclerosis were recruited in each country. Control patients were matched to the patients with multiple sclerosis according to age and sex. Quality of life was assessed using the functional status questionnaire (FSQ). RESULTS: The aspects of QoL that were mostly affected in the three countries under study were physical function and general wellbeing. Social role function decreased with increased severity of disease in France and in particular in Germany. Multiple sclerosis did not seem to have an impact on psychological function. The QoL of control patients was systematically higher than that of patients with multiple sclerosis. CONCLUSIONS: Use of such a generic scale showed that progression of multiple sclerosis is accompanied by a decrease in QoL and suggested that this could be a relevant measurement in assessing the effect of treatment and progression of disease. Variation between countries, however, may be important.

Adult KCNE1-knockout mice exhibit a mild cardiac cellular phenotype.

The KCNE1 gene encodes a channel regulator IsK which in association with the KvLQT1 K+ channel protein determines the slow component of the cardiac delayed rectifier current. We have investigated the cellular electrophysiological characteristics of adult KCNE1-knockout mouse hearts by means of the standard microelectrode technique. Action potential parameters from the ventricular endocardium of KCNE1 -/- mice were indistinguishable from those of KCNE1 +/+ animals. In particular, KCNE1 -/- hearts did not exhibit prolonged repolarization. E-4031, a specific blocker of erg K+ channels consistently prolonged repolarization in KCNE1 +/+ but not in KCNE1 -/- hearts. By contrast, the chromanol compound 293B, a specific blocker of KvLQT1 K+ channel produced comparable effects on repolarization in KCNE1 -/- and KCNE1 +/+ mice. We conclude that invalidation of the mouse KCNE1 gene by homologous recombination leads to a mild cardiac phenotype at the cellular level.

Economic evaluation of multiple sclerosis in the UK, Germany and France.

A cross-sectional cost-of-care study was performed to assess the economic burden of multiple sclerosis (MS) in France, Germany and the UK. Patients were stratified into 3 groups according to the Expanded Disability Severity Scale (EDSS): stages I, II and III, corresponding to mild (EDSS 1.0 to 3.5), moderate (EDSS 4.0 to 6.0) and severe (EDSS 6.5 to 8.0) MS, respectively. 90 patients with MS and 30 non-MS control patients were recruited in each country. Control patients were matched to the patients with MS on the basis of age and gender. Demographic, clinical and economic data during the 3-month period prior to entry were collected in patient interviews. Total costs included actual expenditures, such as direct medical and non-medical costs, as well as indirect costs. From the societal perspective, the total cost of MS for 3 months was estimated at 1,928 US dollars, 3,941 US dollars and 5,678 US dollars in France, 2,772 US dollars, 2,056 dollars and 5701dollars in Germany, and 5,125 US dollars, 6,751 US dollars and 14, 622 US dollars in the UK, for stage I, II and III patients, respectively. The major medical cost driver in the UK was outpatient consultations, whereas hospitalisations were the major component in Germany and France. The major cost in the UK arose from the dependence of patients with MS on caregivers, which caused high non-medical, societal costs compared with France and Germany. From both the societal and health insurance perspectives in each country, costs for control patients were lower than those for stage I MS patients. MS represents a major financial burden on the individual, the family, health services and society, and these costs increase with MS progression.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells.

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the "small-conductance" basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.