Escherichia coli membranes during electrotransformation: an electron microscopy study.

Structural changes undergone by Escherichia coli cell envelope membranes under the conditions of electrically induced gene (DNA) transfer (exponential pulse of about 13 kV/cm, tau = 5 ms) were studied by freeze-fracture electron microscopy. Special device similar to that of Stenger and Hui [1986) J. Membr. Biol. 93, 43-53), that allowed cryofixation of samples almost simultaneously with application of electric pulse, was employed to examine the cells within a short time (less than or equal to 1 s) after the pulse. Extensive blebbing of cells was observed immediately after the pulse. At later times (30-40 s after the pulse) blebbing was not detected, instead infrequent cellular membrane fusion and formation of large membrane 'opening' or pores were observed. An attempt to relate the observed membrane changes with cellular viability and permeability to exogenous DNA failed. Challenge of cells with a plasmid DNA 10 s after the pulse application resulted in a dramatic loss (at least four orders of magnitude) of the number of transformants compared to cells pulsed in the presence of DNA. On the other hand the results on additional pulsing of cell prior to the main electrotransformation procedure suggested that the life-time of membrane defects is at least no less than 2 min. Possible ways to reconcile the results are suggested.

Lipid polymorphism of model and cellular membranes as revealed by electron microscopy.

This review surveys the current state of knowledge relating to lipid polymorphism within both model lipid membrane and cellular membrane systems. Emphasis is placed upon the contribution of data obtained by transmission electron microscopy of freeze-fractured specimens. Some consideration is also given to the other important methods for the study of lipid polymorphism, namely X-ray diffraction and NMR spectroscopy. A detailed account of the different phases found in lipid mixtures within model membranes (bilayer, cubic or tetragonal, tubular or hexagonal) provides the background to the understanding of the factors involved in polymorphic phase transitions. The sequential steps involved in lipid polymorphism are defined from electron microscopical data and are related to the structural changes that can be detected within biological membranes. It is proposed that the fine structural changes detected at the initial stages of polymorphic transition in vivo may be highly relevant in relation to membrane fusion events, to the formation of tight junctions, and even to physiological transport processes. Since the later stages of polymorphic transition generally destroy the permeability barrier of model and cellular membranes, extensive rather than localized phase transition of the lipid bilayer is not at the moment considered to be compatible with cellular viability.

A study of the mechanisms of chloroquine retinopathy. I. Chloroquine effect on lipid peroxidation of retina.

The influence of chloroquine on lipid peroxidation in rat and rabbit retinas has been studied. Chloroquine retinopathy is not followed by an increase in lipid peroxidation, and the antioxidant ionol does not prevent the drug effect. Chloroquine has an inhibitory action on induced lipid peroxidation.

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes.

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.

Model membrane morphology and crosslinking of oxidized lipids with proteins.

Model membranes composed of thion-phosphatidylcholine, cardiolipin, and cytochrome c have been studied by 31P NMR, polyacrylamide gel electrophoresis, gel filtration, fluorescence, and freeze-fracturing. Covalent binding of oxidized phospholipids to cytochrome c was shown to result in the formation of high-molecular-weight oligomeric complexes via Schiff base formation between a protein molecule and aldehydes produced upon peroxidation of phospholipids. The initial steps of the protein oligomerization lead to the appearance of intramembranous particles (IMPs) of various size and distribution on freeze-fractured faces of these model membranes. In the final phase of the crosslinking between cytochrome c and oxidized products of cardiolipin there is a breakdown of membrane vesicles and formation of globular lipoprotein complexes which are seen as globular particles. It is believed that the covalent linking between the products of phospholipid peroxidation and membrane proteins causes the oligomerization of membrane proteins and structural alteration in the hydrophobic region of other models also and, perhaps, in biological membranes.