1-2-dielaidoylphosphatidylethanolamine and 6-carboxyfluorescein

The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.

Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Decorin; Drug Carriers; Extracellular Matrix Proteins; Fibroblasts; Fluoresceins; Lipids; Liposomes; Oligodeoxyribonucleotides, Antisense; Phosphatidylethanolamines; Proteoglycans; Rabbits; Respirovirus; RNA, Messenger; Tissue Distribution

Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational modification, and unitary channel conductance (gamma j). Despite a 10-fold variation in gamma j, whether connexin-specific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that gamma j is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel gamma j (and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2',7'-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na+ and K+ channels), implying similar mechanisms for selectivity.

Topics: Animals; Blotting, Northern; Cells, Cultured; Chickens; Connexins; Diffusion; Electric Conductivity; Electrophysiology; Fluoresceins; Fluorescent Dyes; Gap Junctions; Genetic Vectors; Humans; Ion Channels; Liposomes; Mice; Models, Anatomic; Models, Biological; Molecular Weight; Neuroblastoma; Patch-Clamp Techniques; Phosphatidylethanolamines; Plasmids; Rats; Second Messenger Systems; Surface Properties; Transfection

Among several cholesterol oxidation products, 25-hydroxycholesterol is particularly potent in enhancing basic protein-induced carboxyfluorescence leakage from liposomes. Both myelin basic protein and poly(L-arginine) are effective at submicromolar concentrations in stimulating this vesicle rupture. Leakage is greatly augmented by the presence of only 1% of the 25-hydroxycholesterol. The possible relevance of these findings to myelin pathology is discussed.

Topics: Cholesterol; Fluoresceins; Humans; Hydroxycholesterols; Liposomes; Myelin Basic Protein; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence

A number of sugars lowered the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. Disaccharides had the greatest effect followed by sugar alcohols. The monosaccharides, glucose and galactose had no effect on this phase transition temperature. The sugars promoted vesicle leakage only under conditions where the lipid was near its hexagonal phase transition temperature. Leakage from lipids in the bilayer state was inhibited by the sugars. Polyols, such as sorbitol, promote hexagonal phase formation and alter membrane permeability. These membrane effects may contribute to the damage caused by sorbitol accumulation in certain tissues of diabetic patients.

Topics: Calorimetry, Differential Scanning; Cell Membrane; Diabetes Mellitus; Disaccharides; Fluoresceins; Humans; Lipid Bilayers; Magnetic Resonance Spectroscopy; Phosphatidylethanolamines; Sugar Alcohols; Temperature