Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis

hPAR2 (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys361. We have demonstrated, using autoradiography, that Cys361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR2-expressing cell line. hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR2C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR2 possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR2C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR2, whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR2 expression, agonist sensitivity, desensitization and internalization

MMP-15 Is Upregulated in Preeclampsia, but Does Not Cleave Endoglin to Produce Soluble Endoglin

Preeclampsia is a major pregnancy complication, characterized by severe endothelial dysfunction, hypertension and maternal end-organ damage. Soluble endoglin is an anti-angiogenic protein released from placenta and thought to play a central role in causing the endothelial dysfunction and maternal organ injury seen in severe preeclampsia. We recently reported MMP-14 was the protease producing placentally-derived soluble endoglin by cleaving full-length endoglin present on the syncytiotrophoblast surface. This find identifies a specific drug target for severe preeclampsia; interfering with MMP-14 mediated cleavage of endoglin could decrease soluble endoglin production, ameliorating clinical disease. However, experimental MMP-14 inhibition alone only partially repressed soluble endoglin production, implying other proteases might have a role in producing soluble endoglin. Here we investigated whether MMP-15–phylogenetically the closest MMP relative to MMP-14 with 66% sequence similarity–also cleaves endoglin to produce soluble endoglin. MMP-15 was localized to the syncytiotrophoblast layer of the placenta, the same site where endoglin was localized. Interestingly, it was significantly (p = 0.03) up-regulated in placentas from severe early-onset preeclamptic pregnancies (n = 8) compared to gestationally matched preterm controls (n = 8). However, siRNA knockdown of MMP-15 yielded no significant decrease of soluble endoglin production from either HUVECs or syncytialised BeWo cells in vitro. Importantly, concurrent siRNA knockdown of both MMP-14 and MMP-15 in HUVECS did not yield further decrease in soluble endoglin production compared to MMP-14 siRNA alone. We conclude MMP-15 is up-regulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin

What eddy-covariance measurements tell us about prior land flux errors in CO2-flux inversion schemes

0.2 after 200 km). Separating out the plant functional types did not increase the spatial correlations, except for the deciduous broad-leaved forests. Using the statistics of the flux measurements as a proxy for the statistics of the prior flux errors was shown not to be a viable approach. A statistical model allowed us to upscale the site-level flux error statistics to the coarser spatial and temporal resolutions used in regional or global models. This approach allowed us to quantify how aggregation reduces error variances, while increasing correlations. As an example, for a typical inversion of grid point (300 km × 300 km) monthly fluxes, we found that the prior flux error follows an approximate e-folding correlation length of 500 km only, with correlations from one month to the next as large as 0.6

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72 h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Association between adherence to calcium-channel blocker and statin medications and likelihood of cardiovascular events among US managed care enrollees

<p>Abstract</p> <p>Background</p> <p>Prior studies have found that patients taking single-pill amlodipine/atorvastatin (SPAA) have greater likelihood of adherence at 6 months than those taking 2-pill calcium-channel blocker and statin combinations (CCB/statin). This study examines whether this adherence benefit results in fewer cardiovascular (CV) events.</p> <p>Methods</p> <p>A retrospective cohort study was conducted using administrative claims data from the IMS LifeLink: US Health Plan Claims database, identifying adults already taking CCB or statin (but not both) who had an index event of either initiating treatment with SPAA or adding CCB to statin (or vice versa) between April 1, 2004 to August 31, 2005. Inclusion criteria included age 18+ years, continuously enrolled for minimum of 6 months prior and 18 months following treatment initiation, >1 diagnosis of hypertension, and no prescription claims for SPAA or added CCB or statin for 6 months prior. Exclusion criteria included >1 claim with missing or invalid days supplied, age 65+ years and not enrolled in Medicare Advantage, or history of prior CV events, cancer diagnosis, or chronic renal failure. The primary outcome measure was the rate of CV events (myocardial infarction, heart failure, angina, other ischemic heart disease, stroke, peripheral vascular disease, or revascularization procedure) from 6 to 18 months following index date, analyzed at three levels: 1) all adherent vs. non-adherent patients, 2) SPAA vs. dual-pill patients (regardless of adherence level), and 3) adherent SPAA, adherent dual-pill, and non-adherent SPAA patients vs. non-adherent dual-pill patients.</p> <p>Results</p> <p>Of 1,537 SPAA patients, 56.5% were adherent at 6 months, compared with 21.4% of the 17,910 CCB/statin patients (p < 0.001). Logistic regression found SPAA patients more likely to be adherent (OR = 4.7, p < 0.001) than CCB/statin patients. In Cox proportional hazards models, being adherent to either regimen was associated with significantly lower risk of CV event (HR = 0.77, p = 0.003). A similar effect was seen for SPAA vs. CCB/statin patients (HR = 0.68, p = 0.02). In a combined model, the risk of CV events was significantly lower for adherent CCB/statin patients (HR = 0.79, p = 0.01) and adherent SPAA patients (HR = 0.61, p = 0.03) compared to non-adherent CCB/statin patients.</p> <p>Conclusions</p> <p>Patients receiving SPAA rather than a 2-pill CCB/statin regimen are more likely to be adherent. In turn, adherence to CCB and statin medications is associated with lower risk of CV events in primary prevention patients.</p

Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion

The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

<p>Abstract</p> <p>Background</p> <p>In addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer.</p> <p>Methods</p> <p>We established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (α-thrombin and TFLLR-NH₂). We also quantified NF-κB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of α-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR).</p> <p>Result</p> <p>PAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-κB and EGFR phosphorylation initially were triggered by the activation of PAR1 with α-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-κB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed.</p> <p>Conclusion</p> <p>Our data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-κB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.</p

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

<p>Abstract</p> <p>Background</p> <p>The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).</p> <p>Methods</p> <p>The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.</p> <p>Results</p> <p>We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, <it>CD9 </it>and <it>CALM2 </it>mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.</p> <p>Conclusion</p> <p>This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.</p

Integrated time-lapse geoelectrical imaging of wetland hydrological processes

Wetlands provide crucial habitats, are critical in the global carbon cycle, and act as key biogeochemical and hydrological buffers. The effectiveness of these services is mainly controlled by hydrological processes, which can be highly variable both spatially and temporally due to structural complexity and seasonality. Spatial analysis of 2D geoelectrical monitoring data integrated into the interpretation of conventional hydrological data has been implemented to provide a detailed understanding of hydrological processes in a riparian wetland. This study shows that a combination of processes can define the resistivity signature of the shallow subsurface, highlighting the seasonality of these processes and its corresponding effect on biogeochemical processesthe wetland hydrology. Groundwater exchange between peat and the underlying river terrace deposits, spatially and temporally defined by geoelectrical imaging and verified by point sensor data, highlighted the groundwater dependent nature of the wetland. A 30 % increase in peat resistivity was shown to be caused by a nearly entire exchange of the saturating groundwater. For the first time, we showed that automated interpretation of geoelectrical data can be used to quantify shrink-swell of expandable soils, affecting hydrological parameters, such as, porosity, water storage capacity, and permeability. This study shows that an integrated interpretation of hydrological and geophysical data can significantly improve the understanding of wetland hydrological processes. Potentially, this approach can provide the basis for the evaluation of ecosystem services and may aid in the optimization of wetland management strategies