The sponge phase of a mixed surfactant system.

We study the sponge phase of the mixed non-ionic/ionic surfactant system C14DMAO-TTAB-hexanol-brine. Our aim is to determine if this phase exists in this mixed system and if it preserves or changes its structure when the relative amount of the charged surfactant is increased in the mixture. SAXS, FFEM, and conductivity results show that for the same bilayer volume fraction the sponge phase preserves its global structure.

Fusogenic supramolecular vesicle systems induced by metal ion binding to amphiphilic ligands.

The incorporation of lipophilic ligands into the bilayer membrane of vesicles offers the possibility to induce, upon binding of suitable metal ions, a variety of processes, in particular vesicle aggregation and fusion and generation of vesicle arrays, under the control of specific metal-ligand recognition events. Synthetic bipyridine lipoligands Bn bearing a bipyridine unit as head group were prepared and incorporated into large unilamellar vesicles. The addition of Ni2+ or Co2+ metal ions led to the formation of complexes MBn and MBn2 followed by spontaneous fusion to generate giant multilamellar vesicles.

Biomimetic organization: Octapeptide self-assembly into nanotubes of viral capsid-like dimension.

The controlled self-assembly of complex molecules into well defined hierarchical structures is a promising route for fabricating nanostructures. These nanoscale structures can be realized by naturally occurring proteins such as tobacco mosaic virus, capsid proteins, tubulin, actin, etc. Here, we report a simple alternative method based on self-assembling nanotubes formed by a synthetic therapeutic octapeptide, Lanreotide in water.

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium.

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g.

VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995).

Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes.

We have tested the membrane-protein solubilizing properties of two perfluoroalkylphosphocholines. These compounds belong to a series of fluorinated amphiphiles which are being investigated as potential stabilizing agents for a variety of fluorocarbon-based systems. We are particularly interested in cytochrome b558 from phagocytes, the redox component of NADPH oxidase.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed.

Freeze fracture electron microscopy of lyotropic lipid systems: quantitative analysis of the inverse micellar cubic phase of space group Fd3m (Q227).

An inverse micellar cubic phase of cubic aspect 15 formed by dioleoylglycerol/dioleoylphosphatidylcholine mixtures has been studied by freeze fracture electron microscopy. The structure was well preserved after freezing samples which had been hydrated either in pure water or in 30 vol% aqueous glycerol solutions. Electron microscopy images of high quality and resolution have been obtained.

Polymorphic phase behavior of perfluoroalkylated phosphatidylcholines.

The polymorphic phase behavior of the F-alkyl modified phosphatidylcholines FnCmPC with Fn = CnF2n + 1 and Cm = -(CH2)m- and the physicochemical properties of their aqueous dispersions have been investigated. We show that the supramolecular assemblies formed by F4C4PC, F6C4PC, F8C4PC and F4C10PC dispersed in water consist of liposomes. F6C10PC forms, as does F8C10PC, a ribbon-like phase (two-dimensional centered rectangular lattice) at 25 degrees C, but on heating, it forms a lamellar phase.

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis.

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster eggs during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the form of lipid vesicles during stage 10b. During secretion (stages 10b, 11 and 12) the lipid vesicles are accumulated on the vitelline membrane surface and become flat.

Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum.

PS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti-PS2 labelled peptide of 63,000 Da, corresponding presumably to the mature form of PS2.

Transitional steps in the solubilization of protein-containing membranes and liposomes by nonionic detergent.

Membrane solubilization by dodecyl maltoside was studied, using Ca(2+)-ATPase membranes and liposome preparations as prototypes of biological membranes. In equilibrium dialysis experiments, transition from saturable incorporation of monomeric detergent into the membrane to cooperative binding already occurred at a free detergent concentration about 50% of the cmc. This transition was discontinuous for unilamellar liposomes of dioleoylphosphatidylcholine, but gradual for Ca(2+)-ATPase membranes and multilayered liposomes of sarcoplasmic reticulum lipid.

Freeze-fracture electron microscope study of lipid systems. The cubic phase of space group Pm3n.

The cubic phase Q223 (space group Pm3n) of lipid-water systems has been studied by freeze-fracture electron microscopy. Four types of fracture planes were identified; all display highly ordered two-dimensional domains, each subdivided into subdomains related to each other by displacements and rotations connected to the symmetry of the space group. The images were filtered using cross-correlation averaging techniques and the filtered images were compared with the corresponding planar sections of the electron density map.

Electron microscopic study of supramolecular liquid crystalline polymers formed by molecular-recognition-directed self-assembly from complementary chiral components.

Electron microscopic observation provides insight into the nature of the polymeric supramolecular liquid crystalline species (TP2, TU2)n formed by polyassociation of the complementary components TP2 and TU2 derived from D-, L-, or meso-tartaric acid (where T is any form of tartaric acid, D is the D species, and L is the L species) and from pyridine (P) and uracil (U) derivatives. Increasing the concentration of equimolecular solutions of (LP2 + LU2) mixtures leads to the progressive assembly of very long supramolecular-polymolecular entities. The process involves successively nucleation to give small nuclei, growth to filaments, and lateral association to tree-like species, strings, and fibers.

Interactions between genes involved in exocytotic membrane fusion in paramecium.

Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9a was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the trichocyst membrane to the plasma membrane and in assembly of the "rosette," a complex array of intramembranous particles in the plasma membrane at the trichocyst insertion sites.

Involvement of the protein-protein interactions in the thermodynamics of the electron-transfer process in the reaction centers from Rhodopseudomonas viridis.

Reaction centers from Rhodopseudomonas viridis were reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) liposomes. Freeze-fracture electron micrographs were performed on the samples frozen from temperatures above and below the phase transition temperatures of those lipids (Tc = 23 and 9.5 degrees C, in DMPC and DEPC, respectively).

The egg-shell of Drosophila melanogaster. VI, Structural analysis of the wax layer in laid eggs.

Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 A thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid.

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading).

The crystal lattice of Paramecium trichocysts before and after exocytosis by X-ray diffraction and freeze-fracture electron microscopy.

Paramecium trichocysts are unusual secretory organelles in that: (a) their crystalline contents are built up from a family of low molecular mass acidic proteins; (b) they have a precise, genetically determined shape; and (c) the crystalline trichocyst contents expand rapidly upon exocytosis to give a second, extracellular form which is also an ordered array. We report here the first step of our study of trichocyst structure. We have used a combination of x-ray powder diffraction, freeze-etching, and freeze-fracture electron microscopy of isolated, untreated trichocysts, and density measurements to show that trichocyst contents are indeed protein crystals and to determine the elementary unit cell of both the compact intracellular and the extended extracellular form.

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles.

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e.

Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles.

A study of electrokinetic properties of reconstituted sarcoplasmic reticulum was undertaken to determine the nature of the groups bearing the negative charge of the membrane. After incorporation of phosphatidylcholine into the bilayer, it was found that the Ca2+-ATPase embedded in functional vesicles bore 3e- per mole. When the surface charge density of the hydrodynamic particles became more negatively charged by incorporation of phosphatidylserine molecules, the reconstituted vesicles had a tendency to build large structures resulting from vesicle-vesicle interaction and containing large amounts of divalent cations.

Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation.

Reconstitution of a functional presynaptic membrane possessing calcium-dependent acetylcholine release properties has been achieved. The proteoliposomal membrane obtained gains its acetylcholine-releasing capabilities from presynaptic membrane proteins. At the peak of acetylcholine release, intramembrane particles became more numerous in one of the proteoliposomal membrane faces.

The possible proton translocating activity of the mitochondrial uncoupling protein of brown adipose tissue. Reconstitution studies in liposomes.

Loose coupling of thermogenic mitochondria of brown adipose tissue is related to a high proton (or hydroxyl) conductance of the inner membrane and to the presence of a unique 32 kDa uncoupling protein. Reconstitution experiments of the purified protein in liposomes are reported which suggest that this component could form proton channels in the membrane.

Freeze-fracture electron microscopic and low temperature x-ray scattering studies of the effect of cryofixation upon serum low density lipoprotein structure.

We report here a correlated X-ray diffraction and freeze-fracture electron microscope study of the effects of several cryofixation procedures upon human serum low density lipoprotein (LDL2) structure. Only when the LDL2 solutions contained 75%, by weight, glycerol were the room temperature and post cryofixation low temperature LDL2 X-ray scattering curves indistinguishable from one another. Other cryofixation procedures, slow or rapid, with or without glycerol, resulted in differences between the room temperature and low temperature LDL2 X-ray scattering curves, in part due to the effect of quenching upon the solvent.