Ted by culture in the presence of 700 mg L 1 � �m G418. Non-resistant cells AM-1241 Cannabinoid receptor inhibitor were removed by replacing the cell culture medium for 12 days every 14 days. The only surviving colony was picked and expanded. The protein expression of mouse or human 11b and 11b HSD2 HSD1 was best CONFIRMS, respectively, by Western blot. 11b HSD enzymes were purified, respectively, as previously described. The SPA was used to screen for inhibitors of HSD 11b, wherein the fractions produced by microsomes from transfected HEK 293 cells F Is either human or murine 11b 11b or stable as a source of HSD2 HSD1 enzyme. Briefly, various concentrations of the compound in 96-well microtiter plates were added, followed by the addition of 80 ml of buffer consisting of 50 mM HEPES, pH 7.4, containing 25 nM cortisone and 1.
25 mM NADPH and 12.5 nM cortisol and 0.625 mM NAD. The reactions were terminated by adding 11b or 11b HSD1 HSD2, enzyme preparation fractions of microsomes mg of HEK 293 cells at a final concentration of 80 L 1 initiated � �m HSD1 to 11b and 160 mg L 1 � �m for HSD2 Asiatic acid 464-92-6 11b, respectively. After incubation for 60 at 37, the reaction was quenched by adding 35 stopped ml of 10 mg L � �m a protein A-coated yttrium silicate beads in Superblock blocking buffer with 3 suspended mg � �m L 1 of the murine monoclonal Body cortisol and 314 mM Glycyrrhetins acid. The plates were placed in plastic film on a shaker for 120 min at room temperature incubated before select the Z. The amount of cortisol 11b HSD1 enzyme reaction or the remaining 11b HSD2 enzyme reaction was prepared by the beads detected and determined in a liquid scintillation Counter microplate.
The% inhibition was calculated relative to an uninhibited control. The data were from at least three independent Get ngigen experiments. IC50 values were determined from the concentration-response curves, calculated by nonlinear regression analysis using Prism version 4. DOCK4.0 was used to study the host. The original structure was PDB entry 2IRW and the residues in the vicinity of the ligand in this structure at a radius of 5 Å were isolated from the construction of docking gates. W During the docking calculations, Kollman all atom charges were assigned to the protein, and H Gasterger lid ü loads were assigned to small molecules. Conformational flexibility t of small molecules was For research in the host.
The binding energy of the receptor-ligand was set to approx Hr be the sum of the van and electrostatic interaction energies derWaals. After an initial evaluation of the orientation and scoring, on a minimization of the base of grid was carried out for the ligand to localize to the smallest energy within the local receptor binding site. Position and conformation of each molecule were docked with optimized single anchor search and torsion minimization method. To the activity t to evaluate the acute administration of emodin, C57 BL/6J were administered overnight emodin or vehicle in. Two hours sp ter, the animals were starved get a broken neck tet and the liver and mesenteric fat were immediately isolated, washed in ice-cold PBS, frozen in liquid nitrogen and at 80 Liver and mesenteric fat tissues were fixed in cold homogenization buffer, and 10 mg of liver homogenates or homogenates of 30 mg of mesenteric fat was used to the activity of t in Figure 11b HSD1 PPS analysis, as described above. C57BL/6J m Nnliche Mice were Feeder Llig into six groups on the basis of K Associated with body weight. And experimental groups respectively