1-palmitoyl-2-oleoylphosphatidylcholine and pyranine

A key process of protocell behaviour is their recursive growth and division. In order to be sustainable, the latter must be characterized by an even and homogeneous partition of the solute molecules initially present in the parent protocell among the daughter ones. Here we have investigated, by means of an artificial division model (extrusion of giant lipid vesicles) and confocal microscopy, the fate of solutes when a large vesicle fragments into many smaller vesicles. Solutes of low- and high-molecular weight such as pyranine, calcein, albumin-FITC, dextran-FITC and carbonic anhydrase have been employed. Although the vesicle extrusion brings about a release of their inner content in the environment, the results shown in this initial report indicate that macromolecules can be partially retained when compared with low-molecular weight ones. Results are discussed from the viewpoint of the life cycle of primitive cells. In particular, the findings suggest that a similar mechanism operating during the critical step of vesicle growth-division could have contributed to primitive evolution.

Topics: Albumins; Artificial Cells; Arylsulfonates; Carbonic Anhydrases; Dextrans; Exosomes; Fluorescein-5-isothiocyanate; Fluoresceins; Hydrogen-Ion Concentration; Kinetics; Lipids; Macromolecular Substances; Microscopy, Confocal; Models, Theoretical; Molecular Weight; Phosphatidylcholines; Phospholipids; Stochastic Processes; Synthetic Biology; Systems Biology

We have successfully synthesized a lipid containing the pyranine dye as the hydrophilic headgroup. This lipid was incorporated into liposomes with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine as the major component. The resultant liposomes displayed differential modulations in fluorescence emission intensity in the presence of nanomolar concentrations of different glycosaminoglycans. Linear discriminant analysis of the fluorescence response data demonstrate that the liposomes are able to distinguish between different GAGs. In addition, we also demonstrate that the liposomes incorporating the pyranine lipid are able to distinguish between dilute serum from healthy individuals and serum containing elevated chondroitin sulfate (simulated serum from an Alzheimer's disease patient).

Topics: Arylsulfonates; Chondroitin Sulfates; Discriminant Analysis; Fluorescent Dyes; Glycosaminoglycans; Humans; Liposomes; Phosphatidylcholines; Spectrometry, Fluorescence

The idea that amphotericin B (AmB) may not require sterols to form ion selective channels has recently been criticized on the grounds that egg phospholipids commonly used in experiments may contain small amounts of sterol which associate with AmB to form AmB/sterol pore channel structures. It was recently shown in this laboratory that modest osmotic stress can enhance the formation of AmB channels in sterol-free egg phosphatidylcholine (eggPC) membranes. We have tested AmB's ability to form ion channels/defects in synthetic palmitoyl oleoyl (POPC), dieicosenyl (DEPC) and natural eggPC osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H+ exchange. These sterol-free POPC LUV exhibit greatly increased sensitivity to cation selective AmB channel formation when osmotically stressed; even more than eggPC. Under these stressed conditions, AmB activity was observed at [AmB]/POPC ratios as low as 3.5x10(-4), corresponding to about 34 AmB molecules/vesicle. DEPC vesicles were almost completely unresponsive, demonstrating a strong bilayer thickness dependence. These results prove conclusively that AmB can form sterol-free channels and do so within therapeutic concentration ranges (>0.5-10x10(-6) M) in a stress-dependent manner. This phenomenon may allow us to use osmotic stress changes in simple model systems to spectroscopically isolate and characterize the thus-far elusive AmB channel forming aggregate. In addition, this stress dependence may be responsible for the potentiation of renal toxicity of AmB in the ascending branch of the loop of Henle which is under greatest osmotic stress.

Topics: Amphotericin B; Arylsulfonates; Eicosanoic Acids; Electric Conductivity; Fluorescent Dyes; Ion Channels; Lipid Metabolism; Liposomes; Osmotic Pressure; Phosphatidylcholines