Regulation of epithelial Na+ channel activity by conserved serine/threonine switches within sorting signals

The PY and YXXphi motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the mu(2)-subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na(+) channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)phi). Just preceding the PY and embedded within the YXXphi motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr(628) preceding the PY motif in gamma-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of gamma-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXX motif of gamma-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in gamma-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr(628) and Thr(635) also disrupted ENaC precipitation with the mu(2)-subunit of the AP-2 complex. Moreover, the YXXphi motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXXphi motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.     

A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.     

A role for lymphotoxin in primary Sjogren's disease

The etiology of salivary gland injury in primary Sj?gren's disease is not well understood. We have previously described a mouse model of Sj?gren's disease, IL-14α transgenic (IL14αTG) mice, which reproduces many of the features of the human disease. We now demonstrate a critical role for lymphotoxin α (LTA) in the pathogenesis of Sj?gren's disease in IL14αTG mice. IL14αTG mice express LTA mRNA in their salivary glands and spleen and produce soluble LTA protein in their salivary secretions. When IL14αTG mice were crossed with LTA(-/-) mice, the IL14αTG.LTA(-/-) mice retained normal salivary gland secretions and did not develop either lymphocytic infiltration of their salivary glands or secondary lymphomas. However, both IL14αTG and IL14αTG.LTA(-/-) mice produced similar amounts of IFN-α and had similar deposition of autoantibodies in their salivary glands. Both IL14α and IL14α/LTA(-/-) mice had similar B cell responses to T-dependent and T-independent Ags, L-selectin expression, and expression of RelA, RelB, and NF-κB2 in their spleens. These studies suggest that LTA plays a critical role in the local rather than systemic inflammatory process of Sj?gren's disease. Furthermore, local production of soluble LTA in the salivary glands of IL14αTG mice is necessary for the development of overt Sj?gren's disease. Autoantibody deposition alone is not sufficient to produce salivary gland dysfunction. We also demonstrate that LTA is increased in the salivary gland secretions and sera of patients with Sj?gren's disease, further strengthening the biological relevance of the IL14αTG model to understanding the pathogenesis of human disease.     

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone

Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.     

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers.     

Identification of a noradrenaline-rich subdivision of the human nucleus accumbens

The nucleus accumbens, situated at the junction between rostral pre-commissural caudate and putamen, is now considered to be critically involved in rewarding and motivational functions mediated by the neurotransmitter dopamine. However, in the human, the precise anatomical boundaries of this nucleus are still undetermined and controversy exists as to the extent to which nucleus accumbens activity is controlled by noradrenaline, a related neurotransmitter now much neglected (in favor of dopamine) by the scientific community. Here we resolve the question of noradrenaline in the human nucleus accumbens and identify, in autopsied brain of normal subjects, a small subdivision of the caudomedial portion of this nucleus that selectively contains strikingly high levels of noradrenaline and thus represents the only area in human brain having equally high levels of both noradrenaline and dopamine. The presence of very high, localized noradrenaline concentrations in the caudomedial nucleus accumbens implies a special biological role for this neurotransmitter in human brain motivational processes.     

Voltage-dependent gating underlies loss of ENaC function in Pseudohypoaldosteronism type 1

Here we explore the mechanism and associated structure-function implications of loss of function for epithelial Na⁺ channel (ENaC) containing a pseudohypoaldosteronism type 1 (PHA-1)-causing missense point mutation. As expected, human ENaC that contained subunits harboring PHA-1-causing substitutions within an absolutely conserved, cytosolic Gly residue (e.g., βG37S) had significantly less activity. Unexpectedly, though, such substitution also results in voltage sensitivity with greater activity at hyperpolarizing potentials. This is a consequence of voltage-dependent changes in the single-channel open probability and is not species- or subunit-dependent. Voltage sensitivity in PHA-1 mutants stems from the disruption of critical structure, rather than the development of new properties resulting from the introduction of novel side chains. Residues near the conserved His-Gly sequence of G95 in α-mENaC are particularly important for voltage sensing. Although substitution of I93 in α-mENaC results in voltage sensing, it also slows the activation and deactivation kinetics enough to enable capture of the dynamic changes in single-channel open probability that account for changes in macroscopic activity. This provides definitive proof of the mechanism that underlies loss of function. In addition, the voltage dependence of ENaC with PHA-1 substitutions is akin to that which results from substitution of a critical, interfacial Trp residue conserved at the intracellular base of TM1 (e.g., W112 in α-mENaC). Dynamic interactions between similarly positioned His and Trp residues are essential for gating and the girdle-like structure that lines the intracellular mouth of the M2 proton channel. The similar residues in ENaC may serve a shared function, suggesting the possibility of an intracellular girdle just below the mouth of the ENaC pore.     

Bradykinin acutely inhibits activity of the epithelial Na+ channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).     

Cognitive Distance,Absorptive Capacity and Group Rationality: A Simulation Study

We report the results of a simulation study in which we explore the joint effect of group absorptive capacity (as the average individual rationality of the group members) and cognitive distance (as the distance between the most rational group member and the rest of the group) on the emergence of collective rationality in groups. We start from empirical results reported in the literature on group rationality as collective group level competence and use data on real-life groups of four and five to validate a mathematical model. We then use this mathematical model to predict group level scores from a variety of possible group configurations (varying both in cognitive distance and average individual rationality). Our results show that both group competence and cognitive distance are necessary conditions for emergent group rationality. Group configurations, in which the groups become more rational than the most rational group member, are groups scoring low on cognitive distance and scoring high on absorptive capacity.     

Interactive Local Super-Resolution Reconstruction of Whole-Body MRI Mouse Data: A Pilot Study with Applications to Bone and Kidney Metastases

In small animal imaging studies, when the locations of the micro-structures of interest are unknown a priori, there is a simultaneous need for full-body coverage and high resolution. In MRI, additional requirements to image contrast and acquisition time will often make it impossible to acquire such images directly. Recently, a resolution enhancing post-processing technique called super-resolution reconstruction (SRR) has been demonstrated to improve visualization and localization of micro-structures in small animal MRI by combining multiple low-resolution acquisitions. However, when the field-of-view is large relative to the desired voxel size, solving the SRR problem becomes very expensive, in terms of both memory requirements and computation time. In this paper we introduce a novel local approach to SRR that aims to overcome the computational problems and allow researchers to efficiently explore both global and local characteristics in whole-body small animal MRI. The method integrates state-of-the-art image processing techniques from the areas of articulated atlas-based segmentation, planar reformation, and SRR. A proof-of-concept is provided with two case studies involving CT, BLI, and MRI data of bone and kidney tumors in a mouse model. We show that local SRR-MRI is a computationally efficient complementary imaging modality for the precise characterization of tumor metastases, and that the method provides a feasible high-resolution alternative to conventional MRI.