Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

Effects of the playing surface on plantar pressures during the first serve in tennis.

PURPOSE: This study aimed at examining the influence of different playing surfaces on in-shoe loading patterns in each foot (back and front) separately during the first serve in tennis. METHODS: Ten competitive tennis players completed randomly five first (ie, flat) serves on two different playing surfaces: clay vs GreenSet. Maximum and mean force, peak and mean pressure, mean area, contact area and relative load were recorded by Pedar insoles divided into 9 areas for analysis. RESULTS: Mean pressure was significantly lower (123 ± 30 vs 98 ± 26 kPa; -18.5%; P < .05) on clay than on GreenSet when examining the entire back foot. GreenSet induced higher mean pressures under the medial forefoot, lateral forefoot and hallux of the back foot (+9.9%, +3.5% and +15.9%, respectively; both P < .01) in conjunction with a trend toward higher maximal forces in the back hallux (+15.1%, P = .08). Peak pressures recorded under the central and lateral forefoot (+21.8% and +25.1%; P < .05) of the front foot but also the mean area values measured on the back medial and lateral midfoot were higher (P < .05) on clay. No significant interaction between foot region and playing surface on relative load was found. CONCLUSIONS: It is suggested that in-shoe loading parameters characterizing the first serve in tennis are adjusted according to the ground type surface. A lesser asymmetry in peak (P < .01) and mean (P < .001) pressures between the two feet was found on clay, suggesting a greater need for stability on this surface.

Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors.

Receptor activity modifying proteins RAMP1, RAMP2, and RAMP3 are responsible for defining affinity to ligands of the calcitonin receptor-like receptor (CRLR). It has also been proposed that receptor activity-modifying proteins (RAMP) are molecular chaperones required for CRLR transport to the cell surface. Here, we have studied the respective roles of CRLR and RAMP in transporting CRLR/RAMP heterodimers to the plasma membrane by using a highly specific binding assay that allows quantitative detection of cell surface-expressed CRLR or RAMP in the Xenopus oocytes expression system. We show that: (i) heterodimer assembly is not a prerequisite for efficient cell surface expression of CRLR, (ii) N-glycosylated RAMP2 and RAMP3 are expressed at the cell surface and their transport to the plasma membrane requires N-glycans, (iii) RAMP1 is not N-glycosylated and is transported to the plasma membrane only upon formation of heterodimers with CRLR, and (iv) introduction of N-glycosylation sites in the RAMP1 sequence (D58N/G60S, Y71N, and K103N/P105S) allows cell surface expression of these mutants at levels similar to that of wild-type RAMP1 co-expressed with CRLR. Our data argue against a chaperone function for RAMP and identify the role of N-glycosylation in targeting these molecules to the cell surface.

Direct simulation of interface dynamics: linking capillary pressure, interfacial area and surface energy

We perform direct numerical simulations of drainage by solving Navier- Stokes equations in the pore space and employing the Volume Of Fluid (VOF) method to track the evolution of the fluid-fluid interface. After demonstrating that the method is able to deal with large viscosity contrasts and to model the transition from stable flow to viscous fingering, we focus on the definition of macroscopic capillary pressure. When the fluids are at rest, the difference between inlet and outlet pressures and the difference between the intrinsic phase average pressure coincide with the capillary pressure. However, when the fluids are in motion these quantities are dominated by viscous forces. In this case, only a definition based on the variation of the interfacial energy provides an accurate measure of the macroscopic capillary pressure and allows separating the viscous from the capillary pressure components.

Characterization of a surface metalloprotease from Herpetomonas samuelpessoai and comparison with Leishmania major promastigote surface protease.

The monogenetic kinetoplastid protozoan parasite Herpetomonas samuelpessoai expresses a surface-exposed metalloprotease. Comparable to the Leishmania promastigote surface protease, or PSP, the protease of Herpetomonas is active at the surface of fixed and live organisms, and both enzymes display an identical cleavage specificity toward a nonapeptide substrate. The protease was enriched 440 times by partition into Triton X-114 followed by 2 steps of anion exchange chromatography. The 56-kDa enzyme is inhibited by the metal chelator 1,10-phenanthroline and is susceptible to cleavage by glycosyl-phosphatidylinositol phospholipase C (GPI-PLC). The conservation of an identical surface protease activity in these monogenetic and digenetic trypanosomatids suggests that the enzyme has a physiological function in the promastigote (insect) stage of these parasites.

HLA-C cell surface expression and control of HIV/AIDS correlate with a variant upstream of HLA-C.

A variant 35 kb upstream of the HLA-C gene (-35C/T) was previously shown to associate with HLA-C mRNA expression level and steady-state plasma HIV RNA levels. We genotyped this variant in 1,698 patients of European ancestry with HIV. Individuals with known seroconversion dates were used for disease progression analysis and those with longitudinal viral load data were used for viral load analysis. We further tested cell surface expression of HLA-C in normal donors using an HLA-C-specific antibody. We show that the -35C allele is a proxy for high HLA-C cell surface expression, and that individuals with high-expressing HLA-C alleles progress more slowly to AIDS and control viremia significantly better than individuals with low HLA-C expressing alleles. These data strongly implicate high HLA-C expression levels in more effective control of HIV-1, potentially through better antigen presentation to cytotoxic T lymphocytes or recognition and killing of infected cells by natural killer cells.

A template-assembled synthetic protein surface mimetic of the von Willebrand factor A1 domain inhibits botrocetin-induced platelet aggregation.

Platelet adhesion, the initial step of platelet activation, is mediated by the interaction of von Willebrand factor (VWF) with its platelet receptor, the GPIb-IX complex. The binding of VWF to GPIb-IX is induced either by increased shear stress or by exogenous modulators, such as botrocetin. At a molecular level, this interaction takes place between the A1 domain of VWF and the GPIb alpha chain of the GPIb-IX complex. We report here the design and functional characteristics of a VWF template-assembled synthetic protein (TASP), a chimeric four-helix-bundle TASP scaffold mimicking the surface of the A1 domain. Twelve residues located on helices alpha 3 and alpha 4 in the native A1 domain were grafted onto a surface formed by two neighboring helices of the TASP. VWF TASP was found to inhibit specifically botrocetin-induced platelet aggregation and to bind both botrocetin and GPIb alpha. However, in contrast to the native A1 domain, VWF TASP did not bind simultaneously to both ligands. Modeling studies revealed that the relative orientation of the alpha helices in VWF TASP led to a clash of bound botrocetin and GPIb alpha. These results demonstrate that a chimeric four-helix-bundle TASP as a scaffold offers a suitable surface for presenting crucial residues of the VWF A1 domain; the potential of the TASP approach for de novo protein design and mimicry is thereby illustrated.

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

MOTIVATION: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays.

A surface-based approach to quantify local cortical gyrification.

The high complexity of cortical convolutions in humans is very challenging both for engineers to measure and compare it, and for biologists and physicians to understand it. In this paper, we propose a surface-based method for the quantification of cortical gyrification. Our method uses accurate 3-D cortical reconstruction and computes local measurements of gyrification at thousands of points over the whole cortical surface. The potential of our method to identify and localize precisely gyral abnormalities is illustrated by a clinical study on a group of children affected by 22q11 Deletion Syndrome, compared to control individuals.

The application of trend surface models to the analysis of time factors in Swiss cancer mortality.

To study different temporal components on cancer mortality (age, period and cohort) methods of graphic representation were applied to Swiss mortality data from 1950 to 1984. Maps using continuous slopes ("contour maps") and based on eight tones of grey according to the absolute distribution of rates were used to represent the surfaces defined by the matrix of various age-specific rates. Further, progressively more complex regression surface equations were defined, on the basis of two independent variables (age/cohort) and a dependent one (each age-specific mortality rate). General patterns of trends in cancer mortality were thus identified, permitting definition of important cohort (e.g., upwards for lung and other tobacco-related neoplasms, or downwards for stomach) or period (e.g., downwards for intestines or thyroid cancers) effects, besides the major underlying age component. For most cancer sites, even the lower order (1st to 3rd) models utilised provided excellent fitting, allowing immediate identification of the residuals (e.g., high or low mortality points) as well as estimates of first-order interactions between the three factors, although the parameters of the main effects remained still undetermined. Thus, the method should be essentially used as summary guide to illustrate and understand the general patterns of age, period and cohort effects in (cancer) mortality, although they cannot conceptually solve the inherent problem of identifiability of the three components.

Brain Surface Segmentation of Magnetic Resonance Images of the Fetus

In this work we present a method for the image analysisof Magnetic Resonance Imaging (MRI) of fetuses. Our goalis to segment the brain surface from multiple volumes(axial, coronal and sagittal acquisitions) of a fetus. Tothis end we propose a two-step approach: first, a FiniteGaussian Mixture Model (FGMM) will segment the image into3 classes: brain, non-brain and mixture voxels. Second, aMarkov Random Field scheme will be applied tore-distribute mixture voxels into either brain ornon-brain tissue. Our main contributions are an adaptedenergy computation and an extended neighborhood frommultiple volumes in the MRF step. Preliminary results onfour fetuses of different gestational ages will be shown.

Classification des leucémies aiguës par les marqueurs de surface et corrélation avec le diagnostic morphologique résultats sur 111 cas

111 patients with acute leukemia, including 29 children, were classified according to the surface markers and cytochemistry of their blasts. The acute leukemias were separated into two majors groups (lymphoid and non-lymphoid) depending on the presence or absence of specific lymphoid markers. On the basis of these criteria a correlation of 94% with the hematological diagnosis was obtained. Acute lymphoblastic leukemia (ALL) was divisible into three sub-groups: 11 cases expressing T-cell specific markers were classified as T-ALL and 33 cases expressing the common ALL antigen (CALLA) as c-ALL. 18 of the latter expressed an additional marker, DSA (Daudi surface antigen), splitting c-ALL cases in two subgroups. Cytochemistry of the cases lacking specific surface markers (n = 67) served to diagnose 41 acute myeloid leukemia (AML) cases and 8 monoblastic leukemias. The remaining 18 cases could not be classified. The presence of absence of HLD-DR (Ia) antigens served to subdivide AML into two major subgroups. The prognostic significance of these new diagnostic splits is under active study.