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Studies on transformation of Escherichia coli with plasmids

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Liposomal drug delivery systems: from concept to clinical applications.

Solubilization of membranes by detergents

Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most importantly, a substantial fraction of the aqueous phase (up to 62% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Thus, this relatively simple technique has unique advantages for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents. The preparation and properties of the vesicles are described in detail.

https://www.pnas.org/content/pnas/75/9/4194.full.pdf

Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation

IAbbreviations used in this article are as follows: AraC= l -,B-d arabinofuranosyl cytosine, Chol=cholesterol, DNA=deoxyribonucleic acid, DMPA=dimyristoyl phos­ phatidic acid, DMPC = dimyristoyl phosphatidylcholine, DMPE = dimyristoyl phos­ phatidylethanolamine, DOPC = dioleoyl phosphatidylcholine, DOPE = dioleoyl phos­ phatidylethanolamine, DPPA=dipaJmitoyl phosphatidic acid, DPPC=dipaJmitoyl phos­ phatidylcholine, DPPG = dipaJmitoyl phosphatidylglycerol, DPPS;= dipalmitoyl phos­ phatidylserine, DSPC = distearoyl phosphatidylcholine, EPC = egg phosphatidylcholine, EDTA=ethylene diamine tetracetic acid, HDL=high density lipoprotein, HPLC=high performance liquid chromatography, LUV = large unilamellar vesicle, MLV = multilamellar vesicle, NT A = nitrilotriacetic acid, NMR = nuclear magnetic resonance, PA phosphatidic acid, PC = phosphatidylcholine, PE = phosphatidylethanolamine, PO = phosphatidylglycerol, PS = phosphatidylserine, REV = reverse-phase evaporation vesicle, RNA = ribonucleic acid, SUV=small unilameUar vesicle, Tc=transition temperature. 2Present address: Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.

Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)

Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol.

Differential scanning calorimetry (DSC) and fluorescence polarization of embedded probe molecules were used to detect phase behavior of various phospholipids. The techniques were directly compared for detecting the transition of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) dispersed in aqueous salt solutions. Excellent agreement occurred in the case of phosphatidylcholine; however, in the case of phosphatidic acid, at pH 6.5, transitions detected by fluorescence polarization using the disc-like perylene molecule occurred about 10 degrees lower than those detected by DSC. Discrepancy between fluorescence and DSC methods is eliminated by using a rod-like molecule, diphenylhexatriene (DPH). Both techniques show that doubly ionizing the phosphate group reduces the Tc by about 9 degrees. Direct pH titration of fluidity can be accomplished and this effect is most dramatic when membranes are in their transition temperature range (ca. 50 degrees). Phosphatidic acid transitions occur at higher temperatures, and have appreciably lower transition enthalpies and entropies than phosphatidylcholine. These effect could not be explained simply on the basis of double layer electrostatics and several other factors were discussed in an attempt to rationalize the results. Addition of monovalent cations (0.01-0.5 M) is shown to increase the Tc of dipalmitoylphosphatidylglycerol by less than 3 degrees. However, addition of (1 x 10-3 M) Ca2+ abolishes the phase transition of both phosphatidyglycerol and phosphatidylserine in the range 0-70 degrees. Preliminary X-ray evidence indicates the phosphatidylserine-Ca2+ bilayers are in a crystalline state at 24 degrees. In contrast, 5 x 10-3 M Mg2+ only broadens the transition and increases the Tc indicating a considerable difference between the effects of Ca2+ and Mg2+. Neutralization of PS increases the Tc from 6 degrees (at pH 7.4) to 20-26 degrees (at pH 2.5-3.0) but does not abolish the transition, suggesting the Ca2+ effect involves more than charge neutralization. Addition of Ca2+ to mixed phosphatidylserine-phosphatidylcholine dispersions, induces a phase separation of the dipalmitoyl- (and also distearoyl-) phosphatidylcholine as seen by the appearance of a new endothermic peak at 41 degrees (58 degrees). Similarly, in mixed (dipalmitoyl) phosphatidic acid-phosphatidylcholine (2:1) dispersions, Ca2+ again can separate the phosphatidylcholine component.

Demetrios Papahadjopoulos

Papers

Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)

Phase transitions in phospholipid vesicles. Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol.

Phase transitions and phase separations in phospholipid membranes induced by changes in temperature, pH, and concentration of bivalent cations.

Cochleate lipid cylinders: formation by fusion of unilamellar lipid vesicles.

Effects of Phospholipid Acyl Chain Fluidity, Phase Transitions, and Cholesterol on (Na+ + K+)-stimulated Adenosine Triphosphatase