ABSTRACT
A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Nucleus/metabolism , DNA, Neoplasm/metabolism , Endocytosis , Liver Neoplasms/metabolism , Receptor, Insulin/metabolism , Antigens, Polyomavirus Transforming/metabolism , Biological Transport , Fluorescence , Humans , Microscopy/methods , Plasmids , Polylysine/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
The action of a vital neurotropic dye methylene blue on spontaneous and posttetanic reactions in the autonomic neuromuscular junction was studied with the sucrose gap technique. Low concentrations of methylene blue (10(-5)-10(-6) g/ml) increased the frequency of APs in spontaneously active cells and induced APs in silent cells while high concentrations of methylene blue (10(-3)-10(-4) g/ml) showed the biphasic excitatory-inhibitory action. Similar changes occurred in the excitatory phase of posttetanic response of smooth muscle cells, although the amplitude and duration of synaptic potentials remained unchanged. The stability of reactions after injection of methylene blue simultaneously with ganglio-, adrenoblocking agents and atropine as well as elevated Mg2+, suggests unspecific action of the methylene blue molecule on the muscle cell membrane. The observed phenomena were of shortlasting type and disappeared after washing with normal nutrient medium.
