10 research outputs found
MALDI-TOF MS/MS analysis of the hNAAA tryptic peptide T10-β after covalent modification.
<p>Tandem MALDI-TOF MS/MS spectra of the T10-β peptide (sequence: CTSIVAQDSR) demonstrates covalent modification of Cys126 by both AM6701 (Panel (A)) and <i>N-</i>Cbz-serine β-lactone (Panel (B)).</p
Representation of the active site of hNAAA after treatment with <i>N-</i>Cbz-serine β-lactone.
<p>Homology model illustrates acylated catalytic nucleophile Cys126 after treatment with <i>N-</i>Cbz-serine β-lactone.</p
Mass of tryptic peptide containing Cys126 of hNAAA after covalent modification.
<p>T10-β peptides identified in the tryptic digest of untreated (control) and AM6701 or N-Cbz-serine β-lactone treated hNAAA samples.</p
Potencies of hNAAA inhibitors.
<p>The <i>k</i><sub>inact</sub> and <i>K</i><sub>I</sub> values for the covalent inhibitors were obtained as described in the Experimental Procedures. The IC<sub>50</sub> values were calculated after 2 hours preincubation of the enzyme and inhibitor before addition of the substrate. Values are averages ± SD of three independent experiments.</p
Putative mechanism of inhibition of hNAAA for three compounds studied.
<p>Panel (A). Reversible inhibition of hNAAA by AM9023. Panel (B). Irreversible inhibition of hNAAA by AM6701 via thiocarbamylation of Cys126. Panel (C). Irreversible inhibition of hNAAA by <i>N-</i>Cbz-serine β-lactone most likely proceeds via route 2.</p
Representation of the active site of hNAAA after treatment with AM6701.
<p>Homology model illustrates thiocarbamylation of catalytic nucleophile Cys126 after treatment with AM6701.</p
Concentration dependent inhibition of purified hNAAA by three compounds.
<p>hNAAA was incubated with the compounds AM6701 (squares), <i>N-</i>Cbz-serine β-lactone (circles), and AM9023 (diamonds) for two hours in order to reach full inhibition before measuring activity. Panel (A). A radioactivity-based assay with [<sup>14</sup>C] PEA as substrate. Panel (B). A fluorescence-based assay with PAMCA as substrate. Representative curves are displayed.</p
FAAH mRNA expression in colonic biopsies from patients with IBS-D and IBS-C <i>vs.</i> healthy controls.
<p>The results are shown as mean ± SEM.</p
PEA levels in plasma from IBS patients.
<div><p>PEA levels in plasma 0, 30, 60 and 120 min after blood sample collection from IBS-D (A) and IBS-C (B) patients vs. abdominal cramping frequency.</p>
<p>The results are shown as mean ± SEM of n=7 for each group. Each sample was run in triplicate.</p></div
Endocannabinoid (anandamide, AEA; 2-arachidonoyl glycerol, 2-AG) and cannabinoid-like fatty acid amide (<i>N</i>-oleoyl ethanolamine, OEA; <i>N-</i>palmitoyl ethanolamine, PEA) levels in plasma 0, 30, 60 and 120 min after blood sample collection from IBS-D and IBS-C patients vs.
<div><p><b>healthy controls</b>. </p>
<p>The results are shown as mean ± SEM of n=7 for each group. Each sample was run in triplicate.</p></div