Two adjacent phosphorylation sites in the C-terminus of the channel’s α-subunit have opposing effects on epithelial sodium channel (ENaC) activity

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel’s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to in vitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents.Open Access funding enabled and organized by Projekt DEAL.Friedrich-Alexander-Universität Erlangen-Nürnberg (1041

Regulation of Kir Channels by Intracellular pH and Extracellular K+: Mechanisms of Coupling

ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+o, consistent with coupling between the responses to pHi and K+o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4+ >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+o by altering the properties of the selectivity filter

A mother-child intervention program in adolescent mothers and their children to improve maternal sensitivity, child responsiveness and child development (the TeeMo study): study protocol for a randomized controlled trial

Background: Children of adolescent mothers present a high-risk group for child neglect and maltreatment. Previous findings suggest that early interventions can reduce maltreatment by improving the quality of mother-child interaction, particularly maternal sensitivity. The aim of the current study is to evaluate the effects of a mother-child intervention program using home visits and video-feedback regarding mother-child interaction (STEEP-b) plus psychiatric treatment of the mother in cases where mental illness is present compared with TAU (treatment as usual, that is, standardized support by the child welfare system) on enhancing maternal sensitivity and child responsiveness in adolescent, high-risk mothers. The second aim of the current project is to investigate behavioral and neural differences between adolescent and adult mothers at baseline and postintervention. Methods/Design: This is a randomized controlled trial (RCT) with 120 high-risk adolescent mothers (25 years) will additionally be recruited to investigate behavioral and neural differences between the adolescent and adult group. Blind assessments will take place at T1 (pre-intervention), at the end of the 9-month intervention (T2, postintervention) and 6 months postintervention (T3, follow-up). Moderators of treatment outcomes and sociodemographic data will be assessed at T1. The primary outcome hypothesis is that STEEP-b added to treatment as usual will improve maternal sensitivity and child responsiveness compared with treatment as usual alone in high-risk adolescent mothers. The primary hypothesis will be evaluated at the end of the 9-month follow-up assessment based on the intention-to-treat principle. The trial is funded by the German Ministry for Research and Education (BMBF). Data collection started in October 2012. Discussion: This is a randomized controlled trial that evaluates the effects of an early intervention program (STEEP-b) on the quality of mother-child interaction and child development in adolescent, high-risk mothers. Trial registration DRKS00004409 (27 September 2012

Lack of association of genetic variants in genes of the endocannabinoid system with anorexia nervosa

<p>Abstract</p> <p>Background</p> <p>Several lines of evidence indicate that the central cannabinoid receptor 1 (CNR1) as well as the major endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA) and monoglyceride lipase (MGLL) are implicated in mediating the orexigenic effects of cannabinoids. The aim of this study was to analyse whether nucleotide sequence variations in the <it>CNR1</it>, <it>FAAH</it>, <it>NAAA </it>and <it>MGLL </it>genes are associated with anorexia nervosa (AN).</p> <p>Methods</p> <p>We analysed the association of a previously described (AAT)n repeat in the 3' flanking region of CNR1 as well as a total of 15 single nucleotide polymorphisms (SNPs) representative of regions with restricted haplotype diversity in <it>CNR1</it>, <it>FAAH</it>, <it>NAAA </it>or <it>MGLL </it>in up to 91 German AN trios (patient with AN and both biological parents) using the transmission-disequilibrium-test (TDT). One SNP was additionally analysed in an independent case-control study comprising 113 patients with AN and 178 normal weight controls. Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ARMS-PCR or using 3730xl capillary sequencers.</p> <p>Results</p> <p>The TDT revealed no evidence for association for any of the SNPs or the (AAT)n repeat with AN (all two-sided uncorrected p-values > 0.05). The lowest p-value of 0.11 was detected for the A-allele of the <it>CNR1 </it>SNP rs1049353 for which the transmission rate was 59% (95% confidence interval 47%...70%). Further genotyping of rs1049353 in 113 additional independent patients with AN and 178 normal weight controls could not substantiate the initial trend for association (p = 1.00).</p> <p>Conclusion</p> <p>As we found no evidence for an association of genetic variation in <it>CNR1</it>, <it>FAAH, NAAA and MGLL </it>with AN, we conclude that genetic variations in these genes do not play a major role in the etiology of AN in our study groups.</p

МОДЕЛИРОВАНИЕ ПРОЦЕССОВ ДОСТАВКИ ВСПОМОГАТЕЛЬНыХ ГРУЗОПОТОКОВ ВНУТРИШАХТНОГО ТРАНСПОРТА С УЧЕТОМ ХАРАКТЕРИСТИКИ ТРАССы

Розглянуто специфічні задачі внутрішахтного транспорту. Запропоновано використовувати метод Флойду-Уоршелла для моделювання допоміжних вантажопотоків з урахуванням характеристики мар- шруту. Наведені результати застосування цього методу для своєчасної доставки матеріальних потоків вугільних шахт у підготовчі вибої. Рассмотрены специфические задачи внутришахтного транспорта. Предложено использовать метод Флойда – Уоршелла для моделирования вспомогательных грузопотоков с учетом характеристики трас- сы. Приведены результаты применения используемого метода для оперативной доставки материаль- ных потоков угольных шахт в подготовительные забои

Hemodialysis Patients with Cardiovascular Disease Reveal Increased Tissue Na+ Deposition

Background: The relationship between Na+ balance and cardiovascular disease (CVD) in hemodialysis (HD) patients is not yet fully understood. We hypothesized that HD patients co-diagnosed with CVD show increased tissue Na+ accumulation compared to HD patients without CVD. Methods: In our observational study, 52 HD patients were divided into a group with (23 subjects) or without (29 subjects) a positive history of cardiovascular events. We used 23Na-magnetic resonance imaging (23Na-MRI) at 3.0 Tesla to quantify Na+ content in skin and muscle of both groups directly before and after HD. Additionally, total body fluid distribution was determined by bioimpedance spectroscopy (BIS) and laboratory parameters were assessed. Results: Compared to HD patients without CVD, 23Na-MRI detected an increased Na+ content in skin (21.7 ± 7.3 vs. 30.2 ± 9.8 arbitrary units (a.u.), p + accumulation in HD-CVD patients was paralleled by a higher plasma concentration of the inflammation marker interleukin-6 (5.1, IQR 5.8 vs. 8.5, IQR 7.9 pg/mL, p + content than HD patients without CVD, while no difference in body water distribution could be detected between both groups. Our findings provide evidence that the history of a cardiovascular event is associated with disturbances in tissue Na+ content in HD patients

Fat Mass and Obesity-Associated Gene (FTO) in Eating Disorders: Evidence for Association of the rs9939609 Obesity Risk Allele with Bulimia nervosa and Anorexia nervosa

Objective: The common single nucleotide polymorphism (SNP) rs9939609 in the fat mass and obesity-associated gene (FTO) is associated with obesity. As genetic variants associated with weight regulation might also be implicated in the etiology of eating disorders, we evaluated whether SNP rs9939609 is associated with bulimia nervosa (BN) and anorexia nervosa (AN). Methods: Association of rs9939609 with BN and AN was assessed in 689 patients with AN, 477 patients with BN, 984 healthy non-population-based controls, and 3,951 population-based controls (KORA-S4). Based on the familial and premorbid occurrence of obesity in patients with BN, we hypothesized an association of the obesity risk A-allele with BN. Results: In accordance with our hypothesis, we observed evidence for association of the rs9939609 A-allele with BN when compared to the non-population-based controls (unadjusted odds ratio (OR) = 1.142, one-sided 95% confidence interval (CI) 1.001-infinity; one-sided p = 0.049) and a trend in the population-based controls (OR = 1.124, one-sided 95% CI 0.932-infinity; one-sided p = 0.056). Interestingly, compared to both control groups, we further detected a nominal association of the rs9939609 A-allele to AN (OR = 1.181, 95% CI 1.027-1.359, two-sided p = 0.020 or OR = 1.673, 95% CI 1.101-2.541, two-sided p = 0.015,). Conclusion: Our data suggest that the obesity-predisposing FTO allele might be relevant in both AN and BN. Copyright (C) 2012 S. Karger GmbH, Freibur

Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro

AimsWe explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality.Methods and resultsMurine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm2 in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition.ConclusionBCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair. © 2013 The Author

Tolvaptan treatment in an adult Fontan patient with protein-losing enteropathy: a serial 23Na-MRI investigation

Background: Protein-losing enteropathy (PLE) is a severe complication of the univentricular Fontan circulation and associated with disturbances in salt and water homeostasis. Fontan patients with PLE have a poor prognosis, with increased morbidity and mortality. Due to limited therapeutic strategies, patients are often treated only symptomatically. Methods: We report our first experience of Tolvaptan (TLV) treatment in a Fontan patient with PLE, severe volume retention and hyponatraemia, refractory to conventional diuretic therapy. In addition to clinical parameters, we monitored drug effects including tissue sodium and volume status via serial 23Na-magnetic resonance imaging (23Na-MRI) and bioimpedance spectroscopy compared with age-matched controls. Results: 23Na-MRI identified elevated tissue sodium, which decreased under TLV treatment, as well as volume status, while serum sodium increased and the patient’s symptoms improved. During long-term treatment, we were able to differentiate between sodium and volume status in our patient, suggesting that TLV uncoupled body sodium from water. Conclusion: TLV in addition to loop diuretics improved clinical symptoms of PLE and lowered tissue sodium overload. Long-term effects should be further evaluated in Fontan patients