19 research outputs found
A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells
The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases
The effect of acetic acid extraction time on the dissolution of lead and cadmium in ceramic tiles
With the improvement of people's living standard, the glazed tiles will be decorated with some colored glaze materials in order to increase strength and surface decoration effect, and these ceramic tiles containing lead and cadmium will be dissolved out under the action of acid. The dissolved lead and cadmium and other heavy metals after inhalation will affect the human liver, nervous system, hematopoiesis, and even paralysis. The Influence of detection mechanism and detection time on the results is discussed in this paper
Atomic-Scale Observation of Bimetallic Au-CuOx Nanoparticles and Their Interfaces for Activation of CO Molecules
Supported gold nanoparticles with sizes below 5 nm display attractive catalytic activities for heterogeneous reactions, particularly those promoted by secondary metal (e.g., Cu) because of the well-defined synergy between metal compositions. However, the specific atomic structure at interfaces is less interpreted systematically. In this work, various bimetallic Au-CuOx catalysts with specific surface structures were synthesized and explored by aberration-corrected scanning transmission electron microscopy (AC-STEM), temperature-programmed experiments and in situ DRIFT experiments. Results suggest that the atomic structure and interfaces between gold and CuOx a are determined by the nucleation behaviors of the nanoparticles and result in subsequently the distinctive ability for CO activation. Bimetallic CuO*/Au sample formatted by gold particles surrounded with CuOx nanoclusters have rough surface with prominently exposed low-coordinated Au step defects. Whereas the bimetallic Au@CuO sample formatted by copper precursor in the presence of gold nanoparticles have core-shell structure with relatively smooth surface. The former structure of CuO*/Au displays much accelerated properties for CO adsorption and activation with 90% CO converted to CO2 at 90 degrees C and nice stability with time on stream. The results clearly determine from atomic scale the significance of exposed gold step sites and intrinsic formation of defected surface by different nucleation. The above properties are directly responsible for the induced variation in chemical composition and the catalytic activity
Manganase<sup>2+</sup> stimulation decreases association of ADAM17 to β1 integrin and leads to increased ADAM17 activity.
<p>(A) Control and manganese stimulated (1 mM MnCl<sub>2</sub> in PIPES buffer for 2 min) mesangial cell lysates were immunoprecipitated with ADAM17 antibody. Samples were resolved on a 3–8% Tris-acetate gel and probed for β1 integrin and α5 integrin by Western blotting. Immunoblotting for ADAM17 served as loading control. Arrows point to specific and non-specific (NS) bands. One representative blot out of three is shown. (B) Mesangial cells were transfected with AP-HB-EGF construct and stimulated with 1 mM MnCl<sub>2</sub> in PIPES buffer. HB-EGF shedding was expressed as mean±S.D. of fold increase in the rate of change of relative fluorescence units (RFU); **p<0.01 <i>vs</i> control; n = three experiments, 6 parallels/each condition.</p
ADAM17 co-precipitates with α5β1 integrin and dissociates from the integrin during GPCR stimulation.
<p>(A) Control (−) and 1 µM 5-HT stimulated (+) mesangial cell lysates were immunoprecipitated (IP) with either ADAM17 antibody or Ig control (Ig C), resolved on 3–8% Tris-acetate gel and probed for the presence of β1 integrin and α5 integrin by Western blotting. (B) ADAM17 blot shows that equal amount of ADAM17 were precipitated from each sample. Arrows point to specific and non-specific (NS) bands. One representative example out of four experiments is shown.</p
Cell free assays show ADAM17 binding to α5β1 integrin and changes in ADAM17 activity.
<p>(A) Recombinant ADAM17 binds purified α5β1 integrin and recombinant β1 integrin in a cell free binding assay. Plates pre-coated with antibodies against α5β1 integrin or β1 integrin were incubated with purified α5β1 integrin or with β1 recombinant integrin, individually. Recombinant ADAM17 was then added at the indicated concentrations and ADAM17 binding was measured using a colorimetric assay at 450 nm as described in Methods. Data are expressed as mean±S.D. *p<0.05, **p<0.01 <i>vs</i> control; data from 4 experiments with 3 parallels/each condition are shown. (B) Purified α5β1 integrin and recombinant β1 integrin decrease ADAM17 enzymatic activity. Recombinant ADAM17 (10 ng/ml) was incubated alone or together with either purified α5β1 integrin (25 ng/ml or 250 ng/ml) or with β1 recombinant integrin (12 ng/ml or 120 ng/ml), in OG buffer in the presence of a quenched fluorogenic ADAM17 substrate. Enzyme activity was expressed as the rate of change of relative fluorescence units (ΔRFUs<sup>−1</sup>); *p<0.05, **p<0.01 <i>vs</i> control; data from four experiments with eight parallels/each condition are shown.</p
Co-localization of ADAM17 and α5β1 integrin in rat mesangial cells.
<p>Control (C) and 1 µM 5-HT -stimulated mesangial cell were fixed, permeabilized, and (A) co-immunostained using ADAM17 antibody (green) and β1 integrin antibody (red) as indicated in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033350#s4" target="_blank">Material and Methods</a>”. Arrows indicate co-localization of ADAM17 and α5β1 integrin immunopositive areas (yellow). For the negative controls we omitted the primary antibodies and used PBS followed by secondary antibodies. (B) Parallel samples were incubated with oligonucleotide-labeled PLA probes after incubation with primary antibodies. PLA signals as fluorescence dots were imaged and quantified. As negative control we used either ADAM17 or α5β1 integrin antibody alone followed by the oligonucleotide-labeled PLA probes. Cartoon explains binding of the fluorescence detection reagent only to antibodies in close proximity; **p<0.01. Representative examples out of three experiments are shown.</p
Integrin expression regulates ADAM17 sheddase activity.
<p>(A) Time-dependent release of alkaline phosphatase (AP)-tagged HB-EGF by unstimulated (C) and 5-HT stimulated cells. Cells were transfected with AP-HB-EGF expressing plasmid and 2 days after transfection they were stimulated with 5-HT for the indicated time. AP activity of cell supernatants was determined using Attophos substrate. Data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units; *p<0.05, **p<0.01 <i>vs</i> control at same time point, n = eight experiments, three parallels/each condition. (B) β1 integrin silencing promotes 5-HT-induced AP-HB-EGF shedding. Cells were transfected with AP-HB-EGF expression plasmid together with β1 integrin siRNA (β1-siRNA) or a non-targeting (nt−) siRNA. Successful silencing of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. (C) β1 integrin overexpression inhibits 5-HT induced AP-HB-EGF release in mesangial cells. Cells were transfected with AP-HB-EGF and with β1 integrin expressing plasmid (β1-plasmid) or control DNA (Co-DNA). Successful overexpression of β1 integrin was confirmed by resolving the cell lysates on a 4–12% SDS-PAGE and probing for β1 integrin and β-actin (as loading control). Two days after transfection cells were stimulated with 5-HT for 1 h and AP activity of cell supernatants was determined. Activity data are expressed as mean±S.D. of fold change in the rate of change of relative fluorescence units (RFU); *p<0.05, **p<0.01 <i>vs</i> unstimulated control nt-siRNA or control DNA-transfected cells; <sup>#</sup> p<0.05 and <sup>##</sup> p<0.01 <i>vs</i> 5-HT stimulated nt-siRNA or DNA-transfected cells; n = five experiments, three parallels/each condition.</p
Controlled Synthesis of High-Mobility Atomically Thin Bismuth Oxyselenide Crystals
Non-neutral layered crystals, another
group of two-dimensional (2D) materials that lack a well-defined van
der Waals (vdWs) gap, are those that form strong chemical bonds in-plane
but display weak out-of-plane electrostatic interactions, exhibiting
intriguing properties for the bulk counterpart. However, investigation
of the properties of their atomically thin counterpart are very rare
presumably due to the absence of efficient ways to achieve large-area
high-quality 2D crystals. Here, high-mobility atomically thin Bi<sub>2</sub>O<sub>2</sub>Se, a typical non-neutral layered crystal without
a standard vdWs gap, was synthesized via a facial chemical vapor deposition
(CVD) method, showing excellent controllability for thickness, domain
size, nucleation site, and crystal-phase evolution. Atomically thin,
large single crystals of Bi<sub>2</sub>O<sub>2</sub>Se with lateral
size up to ∼200 μm and thickness down to a bilayer were
obtained. Moreover, optical and electrical properties of the CVD-grown
2D Bi<sub>2</sub>O<sub>2</sub>Se crystals were investigated, displaying
a size-tunable band gap upon thinning and an ultrahigh Hall mobility
of >20000 cm<sup>2</sup> V<sup>–1</sup> s<sup>–1</sup> at 2 K. Our results on the high-mobility 2D Bi<sub>2</sub>O<sub>2</sub>Se semiconductor may activate the synthesis and related fundamental
research of other non-neutral 2D materials
Unraveling the coordination structure-performance relationship in Pt-1/Fe2O3 single-atom catalyst
Heterogeneous single-atom catalyst (SAC) opens a unique entry to establishing structure-performance relationship at the molecular level similar to that in homogeneous catalysis. The challenge lies in manipulating the coordination chemistry of single atoms without changing single-atom dispersion. Here, we develop an efficient synthetic method for SACs by using ethanediamine to chelate Pt cations and then removing the ethanediamine by a rapid thermal treatment (RTT) in inert atmosphere. The coordination chemistry of Pt single atoms on a Fe2O3 support is finely tuned by merely adjusting the RTT temperature. With the decrease in Pt-O coordination number, the oxidation state of Pt decreases, and consequently the hydrogenation activity increases to a record level without loss of chemoselectivity. The tunability of the local coordination chemistry, oxidation states of the metal, and the catalytic performance of single atoms reveals the unique role of SACs as a bridge between heterogeneous and homogeneous catalysis