Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone

AbstractThe meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2–7E). Npr2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events

Bridging the gap between atmospheric concentrations and local ecosystem measurements

This paper demonstrates that atmospheric inversions of CO₂ are a reliable tool for estimating regional fluxes. We compare results of an inversion over 18 days and a 300 x 300 km 2 domain in southwest France against independent measurements of fluxes from aircraft and towers. The inversion used concentration measurements from 2 towers while the independent data included 27 aircraft transects and 5 flux towers. The inversion reduces the mismatch between prior and independent fluxes, improving both spatial and temporal structures. The present mesoscale atmospheric inversion improves by 30% the CO₂ fluxes over distances of few hundreds of km around the atmospheric measurement locations. Citation: Lauvaux, T., et al. (2009), Bridging the gap between atmospheric concentrations and local ecosystem measurements, Geophys. Res. Lett., 36, L19809, doi: 10.1029/2009GL039574

Conversion of the Mycotoxin Patulin to the Less Toxic Desoxypatulinic Acid by the Biocontrol Yeast Rhodosporidium kratochvilovae Strain LS11

Se describe en este artículo el descubrimiento de la degradación de la micotoxina patulina por una levaduraThe infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of 13C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells.

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity

Mesoscale inversion: first results from the CERES campaign with synthetic data

International audienceWe investigate the ability of a mesoscale model to reconstruct CO2 fluxes at regional scale. Formally, we estimate the reduction of error for a CO2 flux inversion at 8 km resolution in the South West of France, during four days of the CarboEurope Regional Experiment (CERES) in spring 2005. Measurements from two towers and two airplanes are available for this campaign. The lagrangian particle dispersion model LPDM was coupled to the non-hydrostatic model Meso-NH and integrated in a matrix inversion framework. Impacts of aircraft and tower measurements are quantified separately and together. We find that the configuration with both towers and aircraft is able to significantly reduce uncertainties on the 4-day averaged CO2 fluxes over about half of the 300×300 km domain. Most of this reduction comes from the tower measurements, even though the impact of aircraft measurements remains noticeable. The noise contributed by imperfect knowledge of boundary inflows does not significantly impair the resolution. We test alternative strategies to improve the impact of aircraft measurements and find that most information comes from measurements inside the boundary layer

Primer sets used for GPR3-HA mutagenesis.

<p>Primer sets used for GPR3-HA mutagenesis.</p

Inhibition of endocytosis increases intracellular cAMP.

<p><b>A)</b> HEK293 cells were co-transfected with GPR3-HA and pcDNA or with Dyn WT or Dyn K44A. Twenty-four hr after transfection, cells were harvested for cAMP EIA. Bars with different letters are significantly different. Significance was determined by One-way ANOVA followed by Newman-Keuls multiple comparison test (<i>p</i><0.01).Results are presented as mean ± S.E.M from 4 separate experiments. <b>B–C</b>) HEK293 cells were co-transfected with GPR3-RFP and CFP-Epac1(δDEP-CD)-YFP and pcDNA, or Dyn WT or Dyn K44A. The YFP/CFP ratios were measured before and after isoproterenol and IBMX treatment. <b>B)</b> Percent change in the YFP/CFP ratio. <b>C</b>) FRET measurements were converted to cAMP levels (µM) as described in the experimental protocol. Bars with different letters are significantly different. Significance was determined by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (<i>p</i><0.01). Results were obtained from 15 different GPR3-RFP expressing cells per group.</p