Precision of farmerbased fertility ratings and soil organic carbon for crop production on a Ferralsol

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Locomotor activation in rats induced by A_2AR antagonists.

<p>Data represent means ± S.E.M. of the locomotor activity (distance traveled, in cm, of total accumulated counts) in habituated rats (90 min) during 90 min following the drug administration (n = 6–8 per group). * and **: p<0.05 and p<0.01, respectively in comparison to vehicle-treated animals (0 mg/kg); ANOVA with <i>post-hoc</i> Newman–Keuls' comparisons, p<0.5 and p<0.01, respectively).</p

Pharmacological parameters for SCH-442416 binding to A_2AR, A₁R-A_2AR and A_2AR-D₂R CHO cells.

<p>Competition experiments of [³H]ZM-241385 (2 nM) binding <i>versus</i> increasing concentrations of SCH-442416 were performed as indicated in Methods in membrane preparations from CHO cells expressing A_2AR or A₁R and A_2AR or A_2AR and D₂R. Results were fitted assuming that receptors (also when heteromerizing) form homodimers, and cooperativity (D_CB ≠ 0, fitting to eq. 2; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016088#s2" target="_blank">Materials and Methods</a>) or non-cooperativity (D_CB = 0, fitting to eq. 3; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016088#s2" target="_blank">Materials and Methods</a>) of SCH-442416 binding was statistically tested (F test). K_DB1 and K_DB2 are, respectively, the equilibrium dissociation constants of the first and second binding of B (SCH-442416) to the dimer. D_CB is the “dimer cooperativity” index for the binding of the ligand B, and B₅₀ is the concentration providing half saturation for B. Data are mean ± S.E.M. values of three experiments.</p><p>**: p<0.01, respectively compared to the K_DB2 and B₅₀ values in A₂R and A₁R-A_2AR cells; Kruskal-Wallis, followed by Dunn's test.</p

Identification of receptor heteromers in CHO cells by BRET saturation curve.

<p>BRET experiments were performed with CHO cells co-expressing A_2AR-<i>RLuc</i> and A₁R-YFP (A) or A_2AR-<i>RLuc</i> and D₂R-YFP (B). Co-transfections were performed with increasing amounts of plasmid–YFP (0.25 to 4 µg cDNA corresponding to A₁R-YFP and 0.5 to 8 µg corresponding to D₂R-YFP) whereas the A_2AR-<i>RLuc</i> construct was maintained constant (0.5 µg cDNA). Both fluorescence and luminiscence of each sample were measured before every experiment to confirm similar donor expressions (about 100,000 luminescent units) while monitoring the increase acceptor expression (10,000–25,000 fluorescent units). As a negative control, linear BRET was obtained in cells expressing equivalent luminescence and fluorescence amounts corresponding to A_2AR-<i>RLuc</i>, (0.5 µg transfected cDNA) and serotonin 5HT_2B-YFP (0.5 to 8 µg transfected cDNA) receptors. The relative amount of acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence value of cells expressing the donor alone (YFP) and the luciferase activity of the donor (Rluc). BRET data are expressed as means ± S.D. of 4–6 different experiments grouped as a function of the amount of BRET acceptor.</p

Blockade by A_2AR antagonists of striatal glutamate release induced by cortical electrical stimulation.

<p>(a) Representative coronal sections of a rat brain, stained with cresyl violet, showing the tracks left by the bipolar stimulation electrode in the orofacial area of the lateral agranular motor cortex (top) and by the microdialysis probe in the lateral striatum (bottom). (b) Effect of systemic administration of the A_2AR antagonists SCH-442416 and KW-6002 (1 mg/kg, i.p., in both cases) on the increase in glutamate extracellular levels in the lateral striatum induced by cortical electrical stimulation. Results are expressed as means ± S.E.M. of percentage of the average of the three values before the stimulation (n = 5–7 per group). Time ‘0’ represents the values of the samples previous to the stimulation. The arrow indicates the time of systemic administration. The train of vertical lines represents the period of cortical stimulation. *: <i>p</i><0.05 compared to value of the last sample before the stimulation (repeated-measures ANOVA followed by Tukey's test).</p

Blockade by A_2AR antagonists of the motor output induced by cortical electrical stimulation.

<p>Dose-dependent decrease in the Power Correlation Coefficient (PCC) induced by the administration of different A_2AR antagonists. Results represent means ± S.E.M. (n = 5–6 per group). * and **: p<0.05 and p<0.01, respectively in comparison to vehicle-treated animals (0 mg/kg); ANOVA with <i>post-hoc</i> Dunnett' comparisons, p<0.5 and p<0.01, respectively).</p

Allosteric interaction between A₁R and A_2AR in A₁R-A_2AR CHO cells.

<p>Competition experiments were performed in membrane preparations from CHO cells expressing A₁R or A₁R and A_2AR with 12 nM [³H]R-PIA <i>versus</i> increasing concentrations of the A_2AR agonist CGS-21680 as indicated in Methods. Data represent means ± S.E.M. of a representative experiment performed with triplicates.</p

Binding of the A_2AR antagonists KW-6002 and SCH-442416 to A₁R-A_2AR and A_2AR-D₂R CHO cells.

<p>Competition experiments of [³H]ZM-241385 (2 nM) <i>versus</i> increasing concentrations of KW-6002 (a and c) or SCH-442416 (b and d) were performed as indicated in Methods in membrane preparations from CHO cells expressing A₁R and A_2AR (a and b) or A_2AR and D₂R (c and d). Data are means ± S.E.M. of a representative experiment performed with triplicates.</p

Allosteric interaction between A_2AR and D₂R in A_2AR-D₂R CHO cells.

<p>Competition experiments were performed in membrane preparations from CHO cells expressing A_2AR and D₂R with 0.5 nM [³H]YM-09151-2 and increasing concentrations of dopamine (from 0.1 nM to 30 µM) in the absence (a) or in the presence (b) of 200 nM CGS-21680 as indicated in Methods. Data represent means ± S.E.M. of a representative experiment performed with triplicates.</p

Functionality of dopamine D₄ receptors in pineal gland and pinealocytes.

<p>Pineal glands extracted at 9:00 h were treated for 10 min with increasing amounts of dopamine or with 1 µM of RO 10-5824 (RO). The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A) and Akt (Ser⁴⁷³) phosphorylation (B), of two separate experiments performed in duplicate were quantified and values represent the mean ± S.D. of the fold increase relative to basal levels found in untreated cells. Significant differences with respect to basal levels were determined by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001). A representative Western blot is shown at the top (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). (C) Pinealocytes were isolated from pineal glands extracted at 9:00 h and were treated with medium (Control), 1 µM of RO 10-5824 (RO), 1 µM phenylephrine (Phenyl), or 1 µM isoproterenol (Iso) for 10 min before labeling with anti-S-arrestin (green) and anti-phospho-ERK1/2 (red), as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cell nuclei were stained with DAPI (blue). Scale bar, 5 µm.</p