PCLS were sonicated in 0.4 ml NaCl (0.9%) before the addition selleck chemical of 2.5 ml of isopropanol/heptane (41) for TG extraction. After 1 h of incubation at room temperature, 1.5 ml of heptane and 1 ml of water were added to the samples. For slices incubated with palmitate, the organic phase was rinsed two times with NaOH 1N/water/ethanol (54550). TG collected from the upper organic phase was counted in 10 ml scintillation fluid (Ultima Gold) in a beta Wallac-1410 counter. For fatty acid oxidation, PCLS were prepared from CT (n=8) and DEF (n=6) fasted mice. After 30 minutes of preincubation, PCLS were incubated in Waymouth medium (supplemented with 0.1 ��M insulin, 1% penicillin/streptomycin and 0.1% bovine serum albumin) containing 0.2 mM [14C]-palmitate (0.4 mCi/mmol) for 3 h at 37��C in a shaking water-bath.

Flasks were gassed with O2/CO2 (955) and sealed with rubber caps equipped with suspended plastic center wells, containing 400 ��l of NaOH 10%. Incubations were terminated by addition of 1 ml of HCLO4 5%. Control flasks were acidified immediately prior to addition of liver slices. To trap the 14CO2, an additional 60 min of shaking was allowed. To determine the amount of produced CO2, all the NaOH was counted in 10 ml scintillation fluid. Hepatic triglycerides secretion Following a 4 h fasting period, CT (n=4) and DEF (n=6) mice were injected with a 10% solution of Tyloxapol (0.5 mg of Tyloxapol/g body weight) (T0307-5G, Sigma-Aldrich, Saint Louis, USA) through the tail vein to inhibit TG degradation by lipoprotein lipase.

Plasma samples were collected through the tail vein before the injection (0 min) and 30, 60, 90 and 120 minutes after Tyloxapol injection. TG in plasma was measured using Diasys Diagnostic and
Hepatitis C virus (HCV), a positive single-stranded RNA virus, is one of the major causes of end-stage liver disease worldwide (5). The nucleotide sequence of the HCV genome is an important predictor of response to antiviral therapy (6). Genotypic analysis is used together with measurement of viral load (VL) to help in the management of patients with HCV infection (1). The 5�� untranslated region (UTR) is often used for monitoring VL because it is less variable than other regions of the genome and consequently less likely to suffer PCR failures due to sequence variation at the primer binding sites (3, 6, 8).

Although this region is not ideal for determination of the genotype, it is often used for this purpose because of the availability of the template after a VL assay and also because the genotypic information needed for clinical use can be obtained from it (2, 4, 9, 10). Our initial attempt to sequence the HCV 5�� UTR using the reverse transcription-PCR Brefeldin_A (RT-PCR) amplicon from the recently introduced Roche Diagnostics (Basel, Switzerland) Cobas AmpliPrep/Cobas TaqMan HCV test (HCV-T) (8) employed a set of sequencing primers originally described by Gargiulo et al.