21) was prepared from pooled plasma of healthy donors and labeled

21) was prepared from pooled plasma of healthy donors and labeled with 3H-cholesteryl ester (PerkinElmer) essentially as described (13), and 1 million dpm of the 3H- cholesteryl ester-HDL were injected via the tail vein into mice that had been given P-407 i.p. 30 min before as detailed above. Blood was drawn 4 h later by heart puncture, TNF-�� inhibitor VLDL was isolated from 200 ��l of plasma as described above, and counts within VLDL were determined by scintillation counting (Beckman LS6500, Palo Alto, CA). Analysis of nascent VLDL composition VLDL (d < 1.006) was isolated from 200 ��l of plasma from each mouse obtained 180 min after P-407 injection as described (14). Enzymatic methods were used to quantitate total cholesterol, free cholesterol, triglycerides, and phospholipids as detailed above.

The bicinchoninic acid method (Pierce, Rockford, IL) was used to quantitate protein. MTP activity assay Assessment of microsomal triglyceride transfer protein (MTP) activity in mouse livers was performed using a fluorigenic assay essentially as described (15, 16). Briefly, to prepare microsomes, mouse liver pieces were washed with PBS, homogenized in 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, 250 mM sucrose, and 0.02% sodium azide using a Polytron homogenizer, and centrifuged (10,900 rpm, 30 min, 4��C; Beckman microcentrifuge). Supernatants were adjusted to pH 5.1 with concentrated HCl, mixed in the cold for 30 min, and centrifuged (13,000 rpm, 30 min, 4��C; Beckman microcentrifuge). Pellets were resuspended in 1 mM Tris-Cl, pH 7.6, 1 mM EGTA, and 1 mM MgCl2, vortexed, incubated for 30 min at 4��C, and ultracentrifuged (50,000 rpm, 4��C, 1 h; SW55 Ti rotor).

Supernatants were used for protein determination and MTP activity measurements in a reaction mixture (100 ��l) containing 3 ��l of donor vesicles [1.2 nmol of phosphatidylcholine (PC) and 100 pmol of fluorescent lipids], 3 ��l of acceptor vesicles (7.2 nmol of PC) in 10 mM Tris, pH 7.4, 0.1% BSA, and 150 mM NaCl buffer. To determine the percentage of lipid transfer, fluorescence values obtained from control assays containing no MTP source (blanks) were subtracted from sample values and then divided by the total fluorescence present in the vesicles reduced by blanks. Analysis of gene expression by real-time quantitative PCR Total RNA from mouse livers was isolated using trizol (Invitrogen), and quantified with a NanoDrop ND-100 UV-Vis spectrophotometer.

cDNA synthesis was performed from 1 AV-951 ��g of total RNA using reagents from Applied Biosystems (Darmstadt, Germany). Real-time quantitative PCR was carried out on an ABI-Prism 7700 (Applied Biosystems) sequence detector with the default settings. PCR primers and fluorogenic probes were designed with the Primer Express Software (Applied Biosystems) and synthesized by Eurogentec (Seraing, Belgium).

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