Search

During transfection, polycation-DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size-structure-transfection activity relationship, we prepared histone HI-DNA complexes in NaCI solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell-cultivation media, the initial structure of H I-DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell-cultivation medium resulted in re-formation and aggregation of the complexes. The addition of the complexes to the cells without cell-cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (corrrelation coefficient 0.91). Small complexes did not contribute to the transfection.

Type 1 diabetes is an autoimmune disease of unknown etiology. Our previous work has shown that a factor present in serum from type 1 diabetic patients causes increased Ca2+ channel activity and apoptotic DNA fragmentation in pancreatic beta-cells. Here we examined the effects of type 1 diabetic serum on primary cerebellar granule cells (CGCs). In CGCs, exposure to type 1 diabetic serum did not cause increased apoptosis or changes in Ca2+ channel activity. However, patient serum did cause modulation of Ca2+ signals in a cell type with triangular soma that exhibited low voltage-gated Ca2+ currents. This cell was present primarily in cultures exposed to type 1 diabetic serum. The presence of low voltage-gated Ca2+ currents and long neuronal dendrites indicated that this unique cell was of neuronal origin and not of glial origin.

A new method has been proposed for analysis of experimental data on ligand-receptor binding at equilibrium. This method makes it possible to detect heterogeneity of a receptor system in cases where the contribution of the high-affinity site to total binding is rather small and the problem of graphic discrimination of a model cannot be solved unambiguously by other methods. The difference method permits us to exclude experiments on measuring nonspecific binding. A computer program for analysis of ligand-receptor binding has been worked out in which the difference method and traditional methods of binding isotherm analysis are realized. Numerical modeling has shown that the best strategy in experimental data processing is the treatment of total binding isotherms by both the difference method and regression analysis, including the nonspecific binding constant as one of the regression parameters.

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.