ABSTRACT
Using lactoferrin as the specific ligand, we developed a simplified method for preparation of molecular conjugate for gene delivery. Replacement of column chromatography and dialysis by one step centrifugal filtration (Centricon, cut off size : 30,000), resulted in the rapid purification of bovine lactoferrin/polylysine (bLf/pL) and human lactoferrin/polylysine (hLf/pL) conjugates and easy separation of unconjugated polylysine. The Lf/pL conjugates prepared by this method efficiently transferred the reporter genes, CAT and LacZ gene, to HeLa and hepatic cells. The bLf/pL and hLf/pL conjugates could transfer the reporter genes to various hepatocytes including primary mouse hepatocyte, Hepa 1-6, SK-Hep1 and Chang liver, but not to NIH 3T3 mouse fibroblast cells, indicating that the Lf/pL conjugates conferred hepatocyte-specific gene transfer. The bLf/pL and hLf/pL conjugates prepared in the present study exhibited higher transfection efficiencies for mouse and human hepatocytes than the commercially available transferrin/polylysine (Tf/pL) conjugate.
Subject(s)
Animals , Cats , Humans , Mice , Chromatography , Dialysis , Fibroblasts , Filtration , Genes, Reporter , Genes, vif , Hepatocytes , Lac Operon , Lactoferrin , Liver , Polylysine , TransfectionABSTRACT
Phenylketonuria is metabolic disorder that results from a deficiency of the hepatic phenylalanine hydroxylase. But among patients with hyperphenylalaninemia, the defect resides in one of the enzymes necessary for production or recycling of tetrahydrobiopterin (BH4). The reduction of BH4 affects not only phenylalanine metabolism but also formation of the tyrosine related neurotransmitter, dopamin and tryptophan related neurotransmitter, serotonin. Administration of L-dopa and 5-hydroxytriptophan seems to be the most effective treatment and may prevent irreversible neurologic damage if started early in life in hyperphenylalaninemia due to deficiency of cofactor BH4. Therefore, all patients with PKU and hyperphenylalaninemia should be tested for BH4 deficiency as early as passible. So we measured reduced forms of biopterin in urines of 19 phenylketonuria patients by Funkushima and Nixon method and 13 of PKU patients measured dihydropterin reductase (DHPR) in white blood cells by modified Narisawa method. We could not find abnormal pterin patterns of cofactor BH4 and normal value of DHPR. All Korean 19 PKU children were classic PKU. A missense mutation has been identified in the phenylalanine hydroxylase (PAH) gene of 16 Koran PKU patients. 5 mutations (IVS4, Y204 C, R243Q, Y356 X, R413 P) have been identified. The frequency of these mutations was found to be 50% of PKU alleles. The IVS4 mutation had a high frequency in Korea and southern China, due to the result of the founder effect and genetic drift. the R413 P mutation, which may have originated in the regions surrounding the Baikal, expanded to northen China and Japan. We were not able to find Caucasian mutations in Korean ptiets. PKU mutations occured after racial divergence between Caucasian and Mongoloids. We observed that PKU patients with Y 204 C and R413 P mutations showed mild mild clinical phenotype but IVS4 mutation had severe mental retardation. the establishment of genotype will therefore aid in the prediction of clinical phenotypes in patients with this disease. So, pterin and DHPR measurement and DNA analsis will be useful for prognosis and proper treatment of PKU patients.