Isotopic evidence of enhanced carbonate dissolution at a coal mine drainage site in Allegheny County, Pennsylvania, USA

18 O SO4 isotopic signatures of the mine drainage and the presence of presumptive SO 4 -reducing bacteria suggest that SO 4 reduction activity also contributes C depleted in 13 C isotope to the total DIC pool. With distance downstream from the mine portal, C isotope signatures in the drainage increased , accompanied by decreased total DIC concentrations and increased pH. These data are consistent with H 2 SO 4 dissolution of carbonate rocks, enhanced by cation exchange, and C release to the atmosphere via CO 2 outgassing

Isotopic evidence of enhanced carbonate dissolution at a coal mine drainage site in Allegheny County, Pennsylvania, USA

18 O SO4 isotopic signatures of the mine drainage and the presence of presumptive SO 4 -reducing bacteria suggest that SO 4 reduction activity also contributes C depleted in 13 C isotope to the total DIC pool. With distance downstream from the mine portal, C isotope signatures in the drainage increased , accompanied by decreased total DIC concentrations and increased pH. These data are consistent with H 2 SO 4 dissolution of carbonate rocks, enhanced by cation exchange, and C release to the atmosphere via CO 2 outgassing

Competitive Regulation of E-Cadherin JuxtaMembrane Domain Degradation by p120-Catenin Binding and Hakai-Mediated Ubiquitination

p120-Catenin binding to, and Hakai-mediated ubiquitination of the E-cadherin juxtamembrane domain (JMD) are thought to be involved in regulating E-cadherin internalization and degradation. However, the relationship between these two pathways is not understood. We targeted the E-cadherin JMD to mitochondria (WT-JMD) to isolate this domain from the plasma membrane and internalization, and to examine protein modifications and degradation. WT-JMD localized to mitochondria, but did not accumulate there except when proteasome activity was inhibited. We found WT-JMD was ubiquitinated, and arginine substitution of lysines at position 5 (K5R) and 83 (K83R) resulted in the stable accumulation of mutant JMD at mitochondria. p120-Catenin did not localize, or bind to WT-JMD even upon proteasome inhibition, whereas the K5,83R-JMD mutant bound and localized p120-catenin to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin binding, but did not decrease JMD stability or its accumulation at mitochondria. Thus, increased stability of JMD lysine mutants was due to inhibition of ubiquitination and not to p120-catenin binding. Finally, mutation of these critical lysines in full length E-cadherin had similar effects on protein stability as WT-JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation

Bioavailability of Macro and Micronutrients Across Global Topsoils: Main Drivers and Global Change Impacts

Understanding the chemical composition of our planet\u27s crust was one of the biggest questions of the 20th century. More than 100 years later, we are still far from understanding the global patterns in the bioavailability and spatial coupling of elements in topsoils worldwide, despite their importance for the productivity and functioning of terrestrial ecosystems. Here, we measured the bioavailability and coupling of thirteen macro- and micronutrients and phytotoxic elements in topsoils (3–8 cm) from a range of terrestrial ecosystems across all continents (∼10,000 observations) and in response to global change manipulations (∼5,000 observations). For this, we incubated between 1 and 4 pairs of anionic and cationic exchange membranes per site for a mean period of 53 days. The most bioavailable elements (Ca, Mg, and K) were also amongst the most abundant in the crust. Patterns of bioavailability were biome-dependent and controlled by soil properties such as pH, organic matter content and texture, plant cover, and climate. However, global change simulations resulted in important alterations in the bioavailability of elements. Elements were highly coupled, and coupling was predictable by the atomic properties of elements, particularly mass, mass to charge ratio, and second ionization energy. Deviations from the predictable coupling-atomic mass relationship were attributed to global change and agriculture. Our work illustrates the tight links between the bioavailability and coupling of topsoil elements and environmental context, human activities, and atomic properties of elements, thus deeply enhancing our integrated understanding of the biogeochemical connections that underlie the productivity and functioning of terrestrial ecosystems in a changing world

Localization and binding of WT-JMD and p120-catenin.

<p>(A) Immunofluorescence of MDCK cells transiently expressing ActA or WT-JMD. Images for RFP (red), p120-catenin (green) and merged are shown separately (100×). Boxed areas are shown as higher magnifications below (RFP-, p120- and merge-inset). All images were from the same experiment and processed identically between cell lines. Scale bar is 25 µm in 100× images, and 5 µm in insets. (B) Lysates and RFP immunoprecipitates (IP) of ActA and WT-JMD stable cell lines under normal conditions, upon proteasome inhibition, or NEM treatment (to inhibit de-ubiquitinating enzymes). Immunoblots (IB) for RFP show: a slower migrating band (upper band-JMD-Ub) that appears only in WT-JMD cells in the presence of MG-132 and NEM; the band identified as ActA/HC comprises a co-migrating ActA and the IgG heavy chain (HC). Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of RFP intensities normalized to tubulin in WT-JMD stables cell lines. Data averaged from 3 independent experiments (+/− s.e.m.), and 2 independently cloned stable cell lines; *p≤0.05, **p≤0.01.</p

E-cadherin JMD is Ubiquitinated.

<p>(A) Lysates of WT-JMD stable cell lines under normal conditions, upon proteasome inhibition (MG-132), or inhibition of deubiquitinating enzymes (NEM). Immunoblot (IB) for RFP shows a slower migrating band (JMD-Ub) in the presence of MG-132 and NEM. (B) In a separate experiment MDCK cells stably expressing WT-JMD were transiently transfected with Ub-HA where indicated. Cells were extracted and RFP immunoprecipitations were preformed under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), addition of the deubiquitinating enzyme Usp2, or mock transfections. DTT was added to the immunoprecipitates after the final was of Protein A beads but prior to the addition of Usp2, where indicated, to neutralize residual NEM. Immunoblots (IB) were performed for RFP using a rabbit polyclonal antibody, followed by a sequential immunoblotting with antibodies specific for: 1) tubulin; and 2) HA. The slowest migrating band (marked as JMD-Ub) that appears in the presence of NEM is positive for RFP and HA (lanes 10 and 11). HC denotes IgG heavy chain. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (C) Extracts from MDCK cells stably expressing WT-JMD were immunoprecipitated (IP) for RFP and E-cadherin under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), or addition of the de-ubiquitinating enzyme Usp2. DTT was added to the immunoprecipitates after the final wash of Protein A beads but prior to the addition of Usp2, where indicated, to neutralize residual NEM. A slow migrating band (lane 15) and protein smear (lanes 11, 13, and 14) appear in E-cadherin immunoprecipitates in the presence of NEM, and collapse upon incubation with Usp2 (similar to RFP immunoprecipitates). RFP immunoblots in lanes 11, 13, 14 & 15 show that the slower migrating band does migrates at different molecular weights indicating variable levels of JMD ubiquitination. Number(s) on the side of the blots are the apparent molecular weights of protein standards (× 10^∧3). Data are representative of 3 different experiments.</p

Lysine mutations inhibit WT-JMD degradation and differentially affect WT-JMD stability.

<p>(A) RFP immunofluorescence of MDCK cells transiently expressing K5R-JMD, K83R-JMD, or K5,83R-JMD under normal conditions (left column), or upon proteasome inhibition (+ MG-132). Images were processed under identical conditions and from the same experiment as images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037476#pone-0037476-g001" target="_blank">Figure 1D</a>. Scale bar is 25 µm. (B) MDCK cells were transiently transfected with ActA or K5,83R-JMD and incubated for 6 hours with cycloheximide, 40 hours post-transfection. Immunoblots (IB) were performed for RFP, β-catenin and GAPDH. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of immunoblots from MDCK cells transiently expressing ActA or K5,83R-JMD and incubated for 6 hours with cycloheximide, 40 hours post-transfection. Band intensities from post nuclear supernatants were normalized to GAPDH to account for reduction in overall protein levels. Results are averaged from 3 independent experiments (+/− s.e.m.).</p

E-cadherin JMD Level is Regulated by Proteasomal Degradation.

<p>(A) Top: schematic representation of the Juxtamembrane domain (JMD) expression construct (left) and ActA control construct (right). Bottom: mouse E-cadherin JMD sequences aligned to demonstrate differences in mutations utilized in this study. Lysine to arginine mutations are denoted by rectangular boxes (K#R and Red “R”). Red AAA denotes mutations to abolish E-cadherin JMD/p120-catenin binding. E-cadherin octapeptide sequence required for binding p120-catenin is underlined. Numbers above the sequence represent the amino acid position number, and the corresponding amino acid position in murine E-cadherin sequence in parenthesis. (B) MDCK cells stably expressing ActA or WT-JMD were incubated for 6 hours with cycloheximide to determine protein turnover. β-catenin used as a degradation control and GAPDH as a loading control. Number(s) on the side of gels represent apparent molecular weights of protein standards (× 10^∧3). (C) Quantification of immunoblots of MDCK cells stably expressing ActA or WT-JMD in cycloheximide time course experiment (see B). Band intensities normalized to GAPDH to account for the reduction in overall protein levels. Results were averaged (+/− standard error of the mean (s.e.m.)) from 3 different experiments (N = 3). (D) Fluorescence images of MDCK cells transiently expressing RFP (ActA) or E-cadherin JMD RFP fusion protein (WT-JMD) targeted to the mitochondria; boxed regions in “RFP” are shown below at a higher magnification in “RFP-inset.” Scale bar is 25 µm in 100X images, and 5 µm in insets. (E) WT-JMD levels increase 4-fold upon proteasome inhibition. WT-JMD migrated with an apparent molecular weight that was ∼15kDa greater than ActA. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (F) Quantification of immunoblot RFP protein levels averaged (+/− s.e.m.) from 3 different experiments; *p≤0.034.</p

Expression of Src-GFP or Hakai-FLAG increase JMD degradation and ubiquitination.

<p>(A) MDCK cells stably expressing WT-JMD were transiently transfected with Src-GFP. Cells were extracted and RFP immunoprecipitations (IP) were performed under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), or addition of the de-ubiquitinating enzyme Usp2. De-ubiquitinating enzyme Usp2 (with DTT) was added, where indicated, to immunoprecipitates after the last step of washing Protein A beads. Membranes were immunoblotted (IB) for RFP, GFP, p120-catenin, and tubulin. Number(s) on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (B) Quantification of immunoblots for WT-JMD upon expression of Src-GFP in WT-JMD stable cells. RFP WT-JMD band intensities from immunoprecipitates were normalized to tubulin to account for reduction in overall protein levels as a result of transfections. *p<0.05. Results are averaged from 3 different experiments (+/− s.e.m.). (C) MDCK cells stably expressing WT-JMD were transiently transfected with Hakai-FLAG. Cells were extracted and RFP immunoprecipitations (IP) were preformed under normal conditions, upon proteasome inhibition, NEM treatment (to inhibit de-ubiquitinating enzymes), or addition of the de-ubiquitinating enzyme Usp2. De-ubiquitinating enzyme Usp2 (with DTT) was added, where indicated, to immunoprecipitates after the last step of washing Protein A beads. Immunoblots (IB) were performed for RFP, p120-catenin and tubulin. Numbers on the side of the gels are the apparent molecular weights of protein standards (× 10^∧3). (D) Quantification of the ratio of JMD-Ub level to total mitochondrial targeted JMD protein (WT-JMD + JMD-Ub) upon expression of Hakai-FLAG in WT-JMD stable cells. Band intensities from immunoprecipitates were normalized to tubulin. Results are averaged from 3 independent experiments (+/− s.e.m.). *p<0.01, **p<0.001.</p

Analysis of full length E-cadherin lysine mutants verifies results of RFP-JMD mitochondrial degradation assay.

<p>(A) Parental MDCK cells and MDCK cells transiently expressing full length E-cadherin RFP fusion proteins (WT-FL), or full length E-cadherin RFP fusion proteins containing lysine mutant sequences (K5R-FL; K83R-FL; K5,83R-FL; AAA-K5,83R-FL). RFP immunoprecipitates were performed 40 hours post-transfection under normal conditions. Immunoblots (IB) for RFP show higher levels of K5R-FL, K5,83R-FL and AAA-K5,83R-FL compared to WT-FL. Immunoblots for p120-catenin show that p120-catenin is co-immunoprecipitated with all E-cadherin mutants, except AAA-K5,83R-FL. (B) Quantification of levels of transiently expressed E-cadherin RFP fusion proteins in MDCK cells. RFP band intensities were normalized to tubulin. Results are averaged from 3–4 different experiments as indicated (+/− s.e.m.); ^∧ p<0.03, *p<0.01, **p<0.001, ***p<0.0001. (C) RFP immunofluorescence of MDCK cells transiently mock transfected, or expressing WT-JMD-FL, K5R-WT-FL, K83R-FL, K5,83R-FL or AAA-K5,83R-FL. All images were taken in cells in which the proteasome was inhibited (+MG-132) to ensure visualization of RFP in WT-JMD-FL and K83R-FL transiently expressing cell lines. Images were taken and processed under identical conditions to each other. Scale bar is 25 µm.</p