Mechanism of ionophoric transport of indium-111 cations through a lipid bilayer membrane.

The use of mobile ionophores to facilitate the transport of 111In through a lipid bilayer membrane has broad applications in liposome technology and cell labeling. However, the mechanism of such ionophore-mediated transport of 111In through a lipid bilayer membrane is not completely clear. The present report describes the correlations of the behaviors of ionophoric loading of 111In into liposomes with the lipophilicity and the indium-binding affinity of three ionophores, namely, 8-hydroxyquinoline, acetylacetone, and tropolone. Our results suggest that the mechanism of the ionophoric transport of 111In through a lipid bilayer membrane involves the rapid exchange of 111In cations among the ionophores in both the aqueous solution and the lipid bilayer. Furthermore, the effectiveness of an ionophore in facilitating the transport of 111In from the external aqueous compartment to the entrapped nitrilotriacetic acid depends not only on the lipophilicity of the [111In]ionophore complex, but also on the lipophilicity of the free ionophore itself and the competition of 111In between nitrilotriacetic acid inside the inner aqueous compartment of the liposome and the ionophore imbedded in the lipid bilayer membrane of the liposome.

Overexpression of HSP70 prevents ultraviolet B-induced apoptosis of a human melanoma cell line.

The heat shock response is a highly conserved reaction common to all cells and organisms. It has been reported that hyperthermic treatment can induce the expression of the heat shock protein (HSP) and can protect cells from ultraviolet (UV) B radiation. In this study, we evaluated the effects of induced HSP70 on resistance to UV radiation. G361 amelanotic human melanoma cells were irradiated with increasing doses of UVB. UVB irradiation caused apoptotic cell death in these cells. Following transfection with MFG.hsp70.puro plasmid, the expression of HSP70 was determined. Compared to control vector-transfected cells, hsp70-transfected cells showed significantly elevated levels of HSP70 and were highly resistant to UVB irradiation. In order to investigate the effects of HSP70 on the apoptotic pathway, the changes in caspase-3 and PARP were analyzed. Following UVB irradiation, activation of caspase-3 and cleavage of PARP were observed in control vector-transfected cells, and the changes in these molecules were inhibited in the hsp70-transfected cells. These results suggest that UVB-induced apoptosis of melanoma cells is accompanied by caspase-3 activation and PARP cleavage, which can be prevented by an overexpression of HSP70.

Application of anion-exchange resin to remove lipophilic chelates from liposomes.

Lipophilic chelates such as 8-hydroxyquinoline, acetylacetone, and tropolone are useful to load high levels of radioactive cations into the inner aqueous compartments of liposomes for investigating the fate of liposomes by the technique of gamma imaging or gamma-ray perturbed angular correlation measurements. However, if lipophilic chelates are not completely removed from liposomes the very same lipophilic chelates can also cause leakage of the entrapped cations from liposomes. Thus, it is essential to make sure that all the lipophilic chelates are removed from liposomes after the loading process. The results of the present study show that more than 99.85% of acetylacetone in liposomal suspension can be removed by a minicolumn of AG1-X8 (phosphate form) anion exchange resin. Virtually all the 8-hydroxyquinoline and tropolone in liposomal suspension are adsorbed tightly to the resin. The procedure is rapid, and the dilution of liposomes is minimal. For experiments involving high levels of gamma-emitting radionuclides, the cleaning up process of removing lipophilic chelates from liposomes can be conveniently operated behind a lead glass.

A nonsense mutation at Arg-1947 in the NF1 gene in a case of neurofibromatosis type 1 in a Korean patient.

We report a case of neurofibromatosis (NF) 1 presenting as a C-to-T transition changing an Arg-1947 codon to a stop codon. Because this mutation has been described in multiple Caucasian and Japanese families, the codon CGA for Arg-1947 in the NF1 gene is considered to be a hotspot for mutation in neurofibromatosis type 1 in all ethnic groups.

Downregulation of Bcl-2 and activation of caspase-8 in the UVB-induced apoptosis of a cultured human melanoma cell line.

PURPOSE: This study was performed to determine the effect of UV radiation on the activation of apoptosis regulatory proteins using cultured human melanoma cells. METHODS: G361 lightly pigmented melanoma cells were irradiated with increasing doses of UVB and analyzed for an apoptotic mechanism using a cell viability test, TEM, FACS, and western blotting analysis. RESULTS: TEM and FACS showed apoptotic features of cell death after UVB irradiation. Western blotting disclosed downregulation of Bcl-2 and the activation of caspase-9. Caspase-8, a downstream molecule of Fas/FasL interaction, was also activated. The activation of downstream molecules of both caspase-8 and caspase-9 was also demonstrated. CONCLUSION: Our data showed that the regulation of the Bcl-2 family and caspase-8 may work together to activate a caspase-3 mediated apoptotic pathway following UVB irradiation of cultured human melanoma cells.

Identification of two novel frame shift mutations of the NF1 gene in Korean patients with neurofibromatosis type 1.

Neurofibromatos is type 1 (NF1) is one of the most common inherited disorders and is characterized by abnormalities in multiple tissues derived from the neural crest. The NF-1 gene has been cloned and mapped to human chromosome 17q11.2. The NF-1 gene has an open reading frame that predicts a protein consisting of 2,818 amino acids, known as neurofibromin. Here, we report two kinds of novel frame shift mutations of the NF1 gene from 2 out of 56 unrelated Korean NF1 patients. These mutations were detected using polymerase chain reaction and single strand conformational polymorphism analysis. Sequencing analysis revealed four base pair insertion at codon 1270 of exon 22, and a base pair deletion at codon 1398 of exon 24. These mutations resulted in premature termination of the mutant alleles and may encode truncated forms of neurofibromin.