Materials and Methods. Beauveriolides I and III were purified from a culture broth of Beauveria sp. FO 6979 as reported. Ls Acid and cholesterol were purchased from DuPont NEN and oleoyl CoA was Pharmacia Amersham Biosciences. DMEM and Hanks balanced Salzl Solution were purchased from Nissui Seiyaku, is GIT medium Nippon SB-715992 CK0238273 Seiyaku, and penicillin, streptomycin and glutamine solutions were from GIBCO L. Phosphatidylcholine, phosphatidylserine, dicetylphosphate, cholesterol, 3-hydroxy-5 pregnene 20 a, Lrot O and free fatty Acid BSA were all from Sigma Aldrich. CL 283,546, an ACAT inhibitor was ? one large generous donation from J. Hess, Pfizer Diagnostics. Plastic microtiter plates were purchased from Corning. Animals.
Female ICR Mice were obtained from Japan SLC, Hamamatsu, BMS-707035 Japan. Low-density lipoprotein knockout M Usen receptor and apolipoprotein E knockout Mice On C57BL 6 background were purchased from The Jackson Laboratory. Mouse peritoneal macrophages. Mouse peritoneal macrophages of female ICR Mice were prepared as described. Peritoneal cells were obtained from unstimulated Mice with Hanks balanced Salzl Solution s, then at 2106 cells per ml in medium harvested GIT. Aliquots were dispensed into 48-well microtiter plates or plastic tissue culture chamber and incubated in a humidified CO 2 at 37 for 2 hours, after which each plate was washed three times with 0.25 ml of Hank’s balanced Salzl Solution to remove cells s only. The medium was subsequently End immediately with 0.
25 ml DMEM serum lipoprotein deficient 8%, penicillin and streptomycin replaced. Determination of Lebensf Ability of the cells. Lebensf Ability of macrophages was measured in the presence of inhibitors with Alamar Blue. Liposomenpr ready. Multilamellar liposomes were prepared as described. Briefly, a lipid mixture of phosphatidylcholine, phosphatidylserine, cholesterol and dicetyl dried in chloroform and then End in 1 ml of 0.3 M glucose. Erg preparation liposomes Complements cholesterol, cholesterol was added to the lipid mixture. Determining the lipid synthesis 14C-labeled neutral by macrophages. Determination of cholesterol esters and triacylglycerol synthesis from Ls Macrophages in acid was performed according to the method described. Briefly, macrophages in a 48 well plate plastic were cultured, then l 2.
5 l sample and 10 l of liposomes containing 5 Ls Acid were added to each culture. After incubation for 14 h, the medium was removed and the cells in each well was washed three times with PBS. The cells were lysed by addition of 0.25 ml of PBS containing 0.1% SDS and cellular Ren lipids were extracted by the method of Bligh and Dyer. The organic L Solvents was reduced by vacuum, were all separated lipids on a TLC plate and analyzed using a Bio-Imaging Analyzer as described. Zellanf coloring Neutral lipids. Macrophages were cultured in a tissue culture room with liposomes and inhibitors as described above. After an incubation of 14 h, the cells were washed three times with PBS and. By immersion in 10% formalin Nuclei and intracellular Re neutral lipid droplets were Tr Matoxylin then with H And L red O found Rbt, and the