APA RESEARCH PAPER

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APA Research Paper

Werner Syndrome

1. Introduction

Werner's syndrome (WS) is a rare human autosomal recessive disorder that mimics many but not all features of premature aging. It is referred to as a segmental progeroid syndrome because it impacts on a number of organ systems and tissues. It is characterized clinically by atherosclerosis, predisposition to cancer, osteoporosis, type 2 diabetes mellitus, and ocular cataracts. The WS gene (WRN) was identified in 1996 by positional cloning and codes for a RecQ helicase (WRN). The human WRN gene and its mouse ortholog Wrn are widely if not ubiquitously expressed. The WRN protein possesses a 30-50 exonuclease activity in addition to its 30-50 helicase activity.

Fifty different mutations of the WRN gene have been reported fromWSpatients and all but tworesult in a truncated proteinwith loss of the nuclear helicase and exonuclease activities. The two exceptions are missense mutations in the exonuclease domain that result in instability of the protein. Although the exact functions of the WRN protein and the molecular deficiencies involved in the clinical phenotype of WS remain unclear, accumulating evidence suggests that it participates in DNA repair, replication, recombination, and telomere maintenance

2. Materials and methods

2.1. Animal care and treatment

All animals used in these experiments were males aged 5-8 weeks. The WrnDhel/Dhel mice have been described. Sprague- Dawley rats and C57BL/6 mice were from Charles River Laboratory (St-Constant, Canada). Animals were treated in accordance with the requirements of the Comite´ de protection des animaux du Centre hospitalier universitaire de Que´bec (Que´ bec, Canada).

2.2. Plasmids

The WRN expression vector contains the full-length murine WRN cDNA cloned into the pCDNA3.1 vector (Invitrogen, Burlington, Canada). Expression vectors for mouse CAR (pCMXmCARb), rat FXR (pCMX-rFXR) and mouse PXR (pCDG-PXR) were from R.G. Evans; those for rat C/EBPa (pCI-rC/EBP) and porcine NF1 (pSK-pNF1) were from L. Belanger; and that for rat LXR (pCMXrLXRb) was from J.-A. Gustafsson.

2.3. RNA isolation and analysis

Wild type and WrnDhel/Dhel C57BL/6 mice were treated with PB (100 mg/kg i.p.) or saline and sacrificed 16 h later. Liverswere rinsed in PBS, pH 7.2 (Invitrogen, Burlington, Canada), frozen in liquid nitrogen and stored at 80 8C until use. Total RNA isolation was performed using TRIzol reagent (Invitrogen) following the supplier's instructions.

2.4. Microsomes and detection of CYP2B proteins by Western blotting

Wild type and WrnDhel/Dhel C57BL/6 mice were treated with PB (100 mg/kg i.p.) or saline and sacrificed 16 h later. Livers were rinsed in cold PBS, frozen in liquid nitrogen and stored at 80 8C until use. Microsomes were isolated and Western blot analysis was performed. Samples of 10 mg of protein per lane were subjected to SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred to PVDF membranes (GE Healthcare) according to the supplier's instructions. After a 1 h incubation in 5% fat-free dried milk in TBS-Tween (20 mM Tris- HCl pH 7.6, 137 mM NaCl, 0.1% Tween 20), CYP2B proteins were detected using a anti-CYP2B1 Ab, a gift of David Waxman, that also recognizes mouse CYP2B ...