No abstract

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptorindependent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide±lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process.To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.Keywords: antennapedia; cellular uptake; dithionite; lipidbinding; structure-activity relationship.The pAntp peptide, a 16 amino-acid segment corresponding to the third helix of the homeodomain of Antennapedia protein is known to be internalized by different cell lines [1±4]. Following conjugation, pAntp has been used as a vector for intracellular delivery of molecules such as peptides [5±8] and oligonucleotides [9,10]. The pAntp peptide is taken up into the cytoplasm and the nucleus of cells both at 37 8C and 4 8C indicating that this internalization does not occur by endocytosis [1]. In addition, the reverso, enantio, and retro-inverso forms of pAntp are also imported into cells demonstrating that the internalization is a receptor-independent process [11,12]. Phosphorus NMR studies show that the interaction between pAntp peptide and a dispersion of brain phospholipid induces the lamellar to inverted hexagonal phase transition [13]. It was therefore proposed that the pAntp peptide interacts with phospholipids and translocates through the bilayer by transient formation of inverted micelles in the membrane. This model implies that the peptide remains in an aqueous environment and may explain why hydrophilic compounds linked to the peptide are tr...

Nuclear translocation of ␤-catenin and its association with Tcf/Lef factors are key steps in transduction of the Wnt signal, which is aberrantly activated in a variety of human cancers. In a search for new ␤-catenin-Tcf target genes, we analyzed ␤-catenin-induced alterations of gene expression in primary human hepatocytes, after transduction of either dominant stable ␤-catenin or its truncated, transactivation-deficient counterpart by means of a lentiviral vector. cDNA microarray analysis revealed a limited set of up-regulated genes, including known Wnt targets such as matrilysin and keratin-1. In this screen, we identified the CXC chemokine interleukin 8 (IL-8) as a direct target of ␤-catenin-Tcf4. IL-8 is constitutively expressed in various cancers, and it has been implicated in tumor progression through its mitogenic, motogenic, and angiogenic activities. The IL-8 promoter contains a unique consensus Tcf/Lef site that is critical for IL-8 activation by ␤-catenin. We show here that the p300 coactivator was required for efficient transactivation of ␤-catenin on this promoter. Ectopic expression of ␤-catenin in hepatoma cells promoted IL-8 secretion, which stimulated endothelial cell migration. These data define IL-8 as a Wnt target and suggest that IL-8 induction by ␤-catenin might be implicated in developmental and tumorigenic processes.

Liver transplantation for liver diseases related to hepatitis B virus (HBV) and hepatitis delta virus (HDV) remains problematic because of the risk of viral recurrence. We report here the long-term virological outcome of patients transplanted for HDV-related liver cirrhosis (HDV cirrhosis). From December 1984 to December 1990, 76 patients with HDV cirrhosis underwent liver transplantation. Before transplantation, all the patients were HBsAg-positive/anti-HDV positive, and all but one were HBV DNA-negative by dot blot hybridization. HDV RNA was detected by HDV RT-PCR and liver HDAg by fluorescent HDV Ab. After transplantation, all the patients except four received continuous long-term anti-HBs passive immunoprophylaxis. The actuarial 5-year survival was 88%. All patients who did not receive anti-HBs immunoprophylaxis remained HBsAg-positive and developed hepatitis. Among the 68 patients receiving antiHBs immunoprophylaxis with a minimum follow-up of 2 months, HBsAg reappeared in 7 (10.3%) after a mean of 17 months. These seven patients developed hepatitis, with simultaneous HBV and HDV replication; and four cleared later HBsAg. Patients without HBV reinfection were studied for HDV reinfection: liver HD Ag or serum HDV RNA were present in 88% of the patients during the first year, without developing hepatitis; however, they were no longer detectable after 2 years in 95% of the patients. In conclusion, liver transplantation for HDV cirrhosis gives good results, with a 5-year actuarial survival of 88%.(ABSTRACT TRUNCATED AT 250 WORDS)