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

Peer Revie

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

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

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

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

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

Higher order complex formation between σ₁ receptors and dopamine D₂ receptors in living cells.

<p>In (<b>a</b>) BRET saturation experiments were performed with HEK-293T cells co-transfected with σ₁-RLuc cDNA (0.2 µg) and increasing amounts of σ₁-YFP cDNA (0.1 to 0.6 µg cDNA). A schematic representation of a BRET process is shown at top in which the receptor fused to RLuc acts as donor and the receptor fused to YFP acts as acceptor. In (<b>b</b>) and (<b>c</b>) SRET saturation experiments were performed with HEK-293T cells co-transfected with: (b) a constant amount of D₂-RLuc (0.6 µg) and D₂-GFP² (1 µg) receptor cDNA (squares) or A_2A-RLuc (0.3 µg) and A_2A-GFP² (0.5 µg) receptor cDNA, as negative control (triangles), and increasing amounts of σ₁-YFP receptor (0.2 to 1.5 µg cDNA), (c) a constant amount of σ₁-Rluc (0.3 µg) and D₂-GFP² (1 µg) (triangles) or A₂-GFP² (0.5 µM) as negative control (squares) receptor cDNA and increasing amounts of σ₁-YFP receptor cDNA (0.2 to 1.5 µg). The relative amount of acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells only expressing the donor, and the luciferase activity of the donor (YFP/Rluc). A schematic representation of a SRET process is shown at top images in which two sequential energy transfer events between Rluc and GFP² (BRET process) and between GFP² and YFP (FRET process) occurs. In (<b>d</b>) BRET with luminescence/fluorescence complementation approach was performed measuring BRET in cells co-transfected with 1 µg of the two cDNAs corresponding to D₂-nRLuc8 and D₂-cRLuc8 and with 1.5 µg of the two cDNAs corresponding to σ₁-nVenus and σ₁-cVenus (5). As negative controls, cells transfected with the same amount of cDNA corresponding to D₂-nRLuc8, D₂-cRLuc8, σ₁-nVenus and cVenus (1), D₂-nRLuc8, D₂-cRLuc8, σ₁-cVenus and nVenus (2), D₂-nRLuc8, σ₁-nVenus, σ₁-cVenus and cRLuc8 (3), or D₂-cRLuc8, σ₁-nVenus, σ₁-cVenus and nRLuc8 (4) did not display any significant luminescence or positive BRET. A schematic representation of a BRET with luminescence/fluorescence complementation approach is given at the top image in which one receptor fused to the N-terminal fragment (nRluc8) and another receptor fused to the C-terminal fragment (cRluc8) of the Rluc8 act as BRET donor after Rluc8 reconstitution by a close receptor-receptor interaction and one receptor fused to an YFP Venus N-terminal fragment (nVenus) and another receptor fused to the YFP Venus C-terminal fragment (cVenus), act as BRET acceptor after YFP Venus reconstitution by a close receptor-receptor interaction. BRET or SRET data are expressed as means ± S.D. of five to six different experiments grouped as a function of the amount of BRET or SRET acceptor.</p