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Summary The present work describes a strategy and the mechanistic background for synthesizing magnetoliposomes (MLs) in which the bi-layered coating is partly composed of positively charged lipids. In a first step, neutral MLs are prepared from zwitterionic phosphatidylcholine vesicles and magnetite nanocores stabilized with an anionic surfactant, and then incubated with cationic vesicles formed from phosphatidylcholine and dioleoyltrimethylammoniumpropane. During this latter step, spontaneous intermembrane lipid transfer occurs. After reaching an equilibrium state, the desired cationic MLs are captured from the mixture in a high-gradient magnetophoresis setup.

Human apolipoprotein A-II (apo A-II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipid-binding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(13-30)-(33-48)-peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix-turn-helix segment spanning residues 13-48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a beta-turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-II-(13-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association.

Background: The cytokeratin (CK) pattern is accepted to be characteristic of a given epithelial cell or tissue. Specific changes in the CK pattern or in the expression of individual CKs may be characteristic in the early development of particular epithelial pathologies. Up to now no systematic hybridohistochemical study on the expression of CKs in normal human esophageal epithelium has been performed. Nevertheless, this knowledge may be of great importance for further research concerning the understanding of the structure and differentiation of normal esophageal epithelium and of the development of non-neoplastic and neoplastic esophageal malignancies. Therefore, we investigated the expression and distribution of nine different CK mRNAs throughout the normal human esophageal mucosa. Methods: A non-radioactive in sity hydridization protocol was used to study the expression of CK mRNAs in fixed and paraffin-embedded human esophageal mucosa. Digoxigenin-labelled cRNA probes were produced by in vitro transcription of cDNA clones, coding for human CKs. Results: In situ hybridization and immunodetection of the hybrids revealed a distinct but different distribution pattern for each specific CK mRNA. The described signal pattern was consistently found at all levels of the esophagus. We observed differences in the expression of some CK mRNAs between the interpapillar and papillar compartment of the esophageal epithelium. Mainly in the papillar regions some mRNAs are already expressed in more basally located cells in comparison with the interpapillar regions. Our results substantiate the hypothesis concerning the formation of papillae in the esophageal mucosa. We have also described some observations on the expression of CK mRNAs in fortuitous sections through excretory ducts of esophageal submucosal glands. Conclusions: The distinct, characteristic, and reproducible distribution pattern observed for each specific CK mRNA indicates that the expression of the genes encoding CKs in the esophageal epithelium as well depends on the cell proliferation, on vertical cell migration and differentiation, and on detachment from the basal lamina. The results presented should be considered as complementary to the already existing immunohistochemical results concerning the distribution of esophageal CK proteins. © 1995 Wiley-Liss, Inc.

We studied the substrate properties of the phospholipid-cholesterol-apolipoprotein complexes generated with apo A-I, apo A-I-CNBr fragments, apo A-II and apo A-IV for cholesterol esterification by the enzyme lecithin-cholesterol acyltransferase (LCAT). The kinetic parameters determined with the different complexes as substrates, showed that the complexes containing apo A-I and apo A-IV were about 40-times more efficient than those generated with the apo A-I fragments. In this system, the substrates containing apo A-II had the lowest efficiency. In spite of the differences in the kinetic parameters observed with the various apolipoprotein-lipid complexes, the cholesterol inserted in the complexes was esterified for more than 90% after 24 h in all systems studied. Based upon the results of the kinetic experiments, we followed the transformation of the discoidal complexes into spherical particles, due to the formation of a cholesteryl esters core, in the presence of low-density lipoproteins as an external source of cholesterol. We observed the formation of spherical particles by electron microscopy, after incubation of the discoidal complexes with LCAT for 24 h. The average percentage of cholesteryl esters in the converted particles was around 60% of the total cholesterol, varying between 40% for the apo A-I-CNBr-1-DPPC-cholesterol complex and up to 86% for the apo A-I-DPPC-cholesterol complex. The secondary structure of protein in the complexes was not significantly modified. However, the phospholipid phase transition disappeared, together with the parallel orientation of the phospholipid acyl chains with the helical segments of the apolipoproteins, as the phospholipids are organized in a monolayer at the surface of the spheres.

The structure, composition, and physico-chemical properties of lipid-protein complexes generated between dimyristoylphosphatidylcholine (DPMC) and the CNBr fragments of human apoA-I were studied. The fragments were separated by high performance liquid chromatography and purified on a reversed-phase column. The complexes with DMPC were isolated on a Superose column; their dimensions were obtained by gradient gel electrophoresis and by electron microscopy. The secondary structure of the protein in the complexes was studied both by circular dichroism and by attenuated total reflection infrared spectroscopy. The fragments 1 and 4 of apoA-I, containing, respectively, two and three amphipathic helices, recombined with the phospholipid to generate discoidal particles with sizes similar to that of apoA-I- and apoA-II-DMPC complexes. The infrared measurements indicated that in all complexes the apolipoprotein helical segments were oriented parallel to the phospholipid acyl chains and that the protein was located around the edges of the discs. Computer modelling of the complexes based on energy minimization techniques proposed a model for these particles in agreement with the dimensions measured experimentally. In conclusion, we propose that apoA-I and its longest CNBr fragments are able to generate discoidal particles with DMPC, with apolipoprotein helical segments oriented parallel to the acyl chains of the phospholipids.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.

The binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to microtubules was analysed by the determination of the concentration of the free enzyme in equilibrium with the complex. At low ionic strength (0.03 M) the binding data are best described by a sum of high (Kd = 0.28 microM) and low affinity (Kd = 7.14 microM) sites, showing positive cooperativity. Addition of 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA) or increasing the ionic strength to 0.1 M reduces the binding constant of the high-affinity sites considerably. Adding microtubule-associated proteins (at I = 0.1 M) does not appreciably influence the affinities. Total stoichiometries vary over 2.1-1.2 tubulin dimers involved in a binding site for GAPDH. Bundling is reduced concomitantly with the reduction of the affinities and the increase of the stoichiometry to close to 1 mol GAPDH/mol tubulin dimer. The critical concentration of tubulin is practically not influenced by the binding of the enzyme. This behaviour is discussed in terms of the concept of linked functions. p[NH]ppA dissociates the bundles very rapidly. Analytical sedimentation studies showed that the dissociation of the bundles by p[NH]ppA is not due to the dissociation of the tetrameric enzyme. Bundling slows down association and dissociation of microtubules. The rate of bundle formation, after addition of GAPDH to preformed microtubules, is not dependent on the GAPDH concentration.

A comparative study that deals with the internalisation of different types of magnetoliposomes (MLs) by 3T3 fibroblasts revealed that cationic MLs proved to be superior to neutral and anionic ones. Internalisation was visualised both by optical light and transmission electron microscopy. The latter showed that the cationic MLs ultimately ended up in lysosomal structures. The effect of increasing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) concentrations in the cationic ML coat has been elucidated. High uptake efficiency was only achieved with MLs that carry a high DOTAP payload. However, these structures also demonstrated toxic effects. The use of the saturated distearoyl analogue (DSTAP) at identical concentrations led to improved uptake efficiency and lower toxicity. By using iron-oxide-free vesicles, it was shown that the toxicity was due to lipid bilayer constituents and not the iron oxide. In conclusion, the use of DMPC-DSTAP (96.67:3.33; molar ratio) MLs results in an extremely high labelling of 3T3 fibroblasts with iron oxides (47.66 pg Fe per cell) without evoking any influence on cell viability.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family. ispartof: FASEB Journal vol:20 issue:3 pages:524-526 ispartof: location:United States status: published

For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin alpha 2 beta 1, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of alpha 2 beta 1 because it only binds to the activated state of alpha 2 beta 1 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of alpha 2 beta 1 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of alpha 2 beta 1 are dependent on the cell type where alpha 2 beta 1 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and alpha 2 beta 1-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of alpha 2 beta 1, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells. Subfaculteit Wetenschappen Campus Kortrijk. Interdisciplinair Research centrum Campus Kortrijk (IRC) Faculteit Geneeskunde. Subfaculteit Geneeskunde Campus Kortrijk. Histologie Campus Kortrijk. ispartof: Journal of Biological Chemistry vol:280 issue:44 pages:36873-36882 ispartof: location:United States status: published

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in the human, where PrPC is converted into an altered form (termed PrPSc). PrPSc is highly resistant towards proteolytic degradation and accumulates in the central nervous system of affected individuals. By analogy with the pathological events occuring during the development of Alzheimer’s disease, controverses still exist regarding the relationship between amyloidogenesis, prion aggregation and neuronal loss. To unravel the mechanism of PrP neurotoxicity and understand the interaction of PrP with cellular membranes, a series of natural and variant peptides spanning residues 118 to 135 of PrP was synthesized. The potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. According to computer modeling calculations, the 120 to 133 domain of PrP is predicted to be a tilted lipid-associating peptide, and to insert in a oblique way into a lipid bilayer through its N-terminal end. In addition to amyloidogenic properties exhibited in vitro by these peptides, peptide-induced vesicle fusion was demonstrated by several techniques, including lipid- and core-mixing assays. Elongation of the 120 to 133 peptide towards the N- and C-terminal ends of the PrP sequence showed that the 118 to 135 PrP peptide has maximal fusogenic properties, while the variant peptides had no effect. Due to their high hydrophobicity, all peptides tested were able to interact with liposomes to induce leakage of encapsulated calcein. We demonstrate also that the propensity of the peptides to fold as an α-helix increases their fusogenic activity, thus accounting for the maximal fusogenic activity of the most stable helix at residues 118 to 135. These data suggest that, by analogy with the C-terminal domain of the β-amyloid peptide, the fusogenic properties exhibited by the prion peptides might contribute to the neurotoxicity of these peptides by destabilizing cellular membranes.

It has recently been shown that triclosan protects the human skin from the inflammation that may be caused by exposure to sodium lauryl sulphate (SLS). The aim of the present study was to examine whether triclosan can protect the hamster cheek pouch mucosa from the irritation caused by exposure to SLS. After four daily applications of a paste containing SLS, the epithelium of the hamster cheek pouch showed consistently prominent structural changes, especially basal hyperplasia, acanthosis, hypergranulosis, and hyperkeratosis. Identical morphological changes were also observed after applications of a paste containing SLS together with triclosan. In contrast, after applications of a paste containing triclosan alone, the cheek pouch mucosa revealed a histological structure essentially similar to the non-treated control mucosa. From these results, we may conclude that SLS, but not triclosan, irritates the hamster cheek pouch epithelium. Moreover, triclosan does not protect the cheek pouch mucosa against structural changes induced by SLS. It must be taken into account that triclosan does not always offer protection against the side-effects of SLS.

The structure, composition and physico-chemical properties of complexes generated between phospholipids and synthetic model peptides for the amphipathic helices of the plasma apolipoproteins were studied. The sequences of the peptides were derived from that of the 18A peptide and designed to either enhance or decrease ionic interactions between pairs of peptides, as described in the accompanying paper. Complexes were prepared with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or with DPPC and cholesterol, and isolated on a Superose 6HR column. Association kinetics for the DMPC-peptides complexes were followed by measuring the turbidity as a function of the temperature. The diameters of the DPPC-peptide complexes, measured by gradient gel electrophoresis (GGE), were about 120 A. Fluorescence polarization measurements after labeling with diphenyl hexatriene (DPH) yielded transition temperatures of, respectively, 40.6, 41.5 and 41.8 degrees C for the DPPC/18AM1-, DPPC/18AM4- and DPPC/18A-peptide complexes. These values were confirmed by differential scanning calorimetry. Circular dichroism and infrared spectroscopy revealed that the peptides adopt an alpha-helical structure in solution and this percentage increased from 30-40% in the free peptides up to 50-60% in the complexes. Attenuated total reflection (ATR) infrared measurements of the complexes indicated that the peptides are oriented parallel to the acyl chains of the phospholipid bilayer. Denaturation of the peptides and of the peptide-lipid complexes was monitored by Trp fluorescence under addition of increasing amounts of GdmCl. The mid-points of the denaturation curves lie at, respectively, 0.05, 0.25 and 0.35 M GdmCl for the 18AM4, 18A and 18AM1 peptide and are shifted towards higher GdmCl concentrations after peptide-lipid binding. GdmCl denaturation decreased the alpha-helical content of the peptides and of the complexes, as monitored by circular dichroism measurement. The helix to random coil structure transition occurred at, respectively, 2.1, 2.2, and 2.0 M GdmCl for 18A, 18AM1 and 18AM4, compared to 5.1, 5.0, and 5.3 M in the corresponding complexes. These data suggest altogether that the structural properties, the mode of lipid-protein association and the stability of the phospholipid-peptide complexes are similar to those of native plasma apolipoproteins. The 18A and 18AM4 peptides which contain charged residues along the edge of the helix, leading to salt bridge formation between peptides were shown to mimic the amphipathic helices of the plasma apolipoproteins.

In the present study, the presence of immunoglobulins was immunohistochemically investigated in globule leucocytes in the tracheal respiratory epithelium of conventional rats, athymic nude rats and a germ-free rat. It was not possible to demonstrate any immunoglobulins in tracheal globule leucocytes. This and other dissimilarities with intestinal globule leucocytes are briefly discussed. On the other hand, peroxidase-antiperoxidase staining revealed the presence of surface immunoglobulins on the membrane of subepithelial mast cells. The previously postulated mast cell origin of the tracheal globule leucocytes is doubted, and the possibility that globule leucocytes should belong to the group of natural killer cells is discussed.

In this cytochemical study, the presence of acid phosphatase and alkaline phosphatase was investigated in globule leucocytes isolated from rat tracheal epithelium. The isolation and enrichment procedure used yields a cell fraction of apparently undamaged globule leucocytes, exhibiting a cell viability of at least 96% as assessed by trypan blue exclusion, and surviving at least in short-term culture. The cytochemical methods applied demonstrate that isolated tracheal globule leucocytes are positive for acid phosphatase activity, but negative for alkaline phosphatase activity. The acid phosphatase acitivity is fluoride-sensitive, as it is completely inhibited in the presence of 10 mM NaF. Isolated rat peritoneal cells were used as positive control cells for the cytochemical reactions.The possible significance of acid phosphatase in globule leucocytes is discussed in connection with the previously suggested cell functions.

We investigated the role of the nature of the casein micelle surface, as affected by adsorption of heat-denatured whey proteins, on yogurt texture. Depending on the heating process and heat intensity, a proportion of β- lactoglobulin and α-lactalbumin bound to the casein. As shown by electron microscopy, filaments of denatured β-lactoglobulin developed on the micellar surface and offered a barrier to micelle fusion when milk was fermented, resulting in inferior yogurt texture. At higher heat intensities, α-lactalbumin also commenced to precipitate onto the micelle, which apparently filled up the gaps formed by the β-lactoglobulin filaments and caused a smoother micellar surface. During fermentation, coalescence of the micelles improved texture. The heat-induced association of α-lactalbumin with the casein particles appeared to be important for fusion and hydration of the micelles during subsequent fermentation, which determined the Theological properties of yogurt.

Several and various types of cells contain fine cytoplasmic filaments closely resembling the myofilaments of muscle cells (2, 18, 23, 24). In many of these cells and especially when cultured, it has been demonstrated that some of these filaments react with heavy meromyosin (HMM) in the same way as do the actin filaments of muscle cells (3, 6 7). This suggests that these filaments may be actinoid and form part of a contractile system. As fine intracytoplasmic filaments do occur in lymphatic endothelial cells (2, 14), we undertook an electron microscope investigation of their fine structure and their reaction on incubation with HMM and EDTA. We postulated that lymphatic endothelial cells possess a contractile filamentous system to which these filaments belong.

The enzyme naphthol-AS-D-chloroacetate esterase is histochemically demonstrated in the granules of globule leucocytes in the tracheal epithelium of rats. The enzyme reactivity may be used as a cytochemical marker of these cells. The previously postulated mast cell origin of globule leucocytes is doubted, and the possibility that globule leucocytes belong to the group of natural killer cells is discussed. The biological role of the located esterase and the function of the globule leucocytes are also briefly discussed.

Globule leukocytes, dispersed throughout the respiratory epithelium as single cells, were isolated from rat trachea and were enriched by centrifugation on a discontinuous Percoll gradient. The isolation and enrichment procedure yields a cell fraction containing 75% globule leukocytes. The cell viability, as assessed by trypan blue exclusion, was at least 98%. Cells were maintained in short-term culture without apparent loss of viability and enzyme activity. The isolated globule leukocytes seem not to express significant levels of cytotoxicity against 51Cr-labeled YAC-1 target cells. In fixed cytocentrifuge smears, globule leukocytes appear as more or less rounded to oval cells with a low nucleo-cytoplasmic ratio and with a mostly eccentrically located nucleus. Characteristic intracytoplasmic granules are stained with toluidine blue, alcian blue, and May-Grunwald-Giemsa stains. The applied cytochemical methods demonstrate that tracheal globule leukocytes are stained for alpha naphthyl acetate esterase and naphthol-AS-D-chloroacetate esterase, but not for alpha naphthyl butyrate esterase, N-acetyl-DL-phenylalanine-B-naphthyl esterase, and endogenous peroxidase. Isolated rat peritoneal cells were used as positive control cells for the cytochemical reactions. The obtained cytochemical profile of tracheal globule leukocytes is compared to the known cytochemical profile of intestinal globule leukocytes and large granular lymphocytes. The cytochemical dissimilarities between tracheal and intestinal globule leukocytes may suggest that both kinds of globule leukocytes represent a different form of the same cell type or even different cell types. The cytochemical pattern of tracheal globule leukocytes is closely related to that of large granular lymphocytes, which have been postulated as a possible source for globule leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Globule leucocytes are described in the tracheal epithelium of young and adult rats. They do not appear in newborn rats up to 4 weeks. Globule leucocytes migrate through the epithelium to be found free in the tracheal lumen. They seem to be retarded a certain time in the apical part of the epithelium. The inclusions of the globule leucocytes are divided into four types. The different steps in the formation and transformation process of these inclusions are suggested. The electron-dense content of the inclusions is digested by pepsin, indicating that they contain protein. This result is not conflicting with the opinion that the globule leucocytes should contain immunoglobulins.

The ultrastructure of globule leukocytes is described in the nasal and tracheal respiratory epithelium of three boys suffering from chronic airway infections. The globule leukocytes lie free in the intercellular spaces and appear to be migratory cells. They are characterized by intracytoplasmic membrane-bound globules, variable in number, size, shape, and internal structure. The globules may apparently release their content between the neighboring epithelial cells. Human globule leukocytes are also characterized by the presence of intracytoplasmic rod-shaped bodies, the significance of which is not known. They usually display an extended juxta-nuclear Golgi apparatus, presumably involved in the formation of the globules. Comparison of the fine structure of the globules in the globule leukocytes with that of the granules found in the subepithelial mast cells does not support a mast cell origin for human globule leukocytes. On morphological grounds, natural killer cells are postulated as a possible source for globule leukocytes. The function of globule leukocytes is briefly discussed. We presume that the globule leukocytes belong to the group of migrating and secreting cells involved in the defense of the organism against foreign material.

The bronchiolar Clara cells of rats contain characteristic rod-shaped granules always surrounded by a unit membrane. These granules contain thin filaments about 9 to 10 nm in diameter lying in a pale matrix. Our morphological results suggest that the filamentary rod-shaped granules originate from the common, round-to-oval, electron-dense Clara cell granules, as we found different intermediate structures between these two kinds of granules. The electron-dense granules are digested by pepsin, whereas the filamentary rod-shaped granules are apparently not affected. The biochemical nature and the possible function of the filamentary rod-shaped granules are also discussed in relation to the secretory activity of the Clara cells.

Nous avons etudie les mitochondries dans les cellules endotheliales de lymphatiques pulmonaires chez des lapins nouveau-nes apres instillation intratracheale de ferritine ou de charbon. Les mitochondries des cellules qui ont endocyte de la ferritine et qui ont ete fixees au glutaraldehyde et au tetroxide d'osmium contiennent apres coloration a l'acetate d'uranyle et au citrate de plomb des aggregations de granules plus ou moins rondes (diametre de 300 a 800 A) qui d'apres nous, n'ont pas encore ete decrites auparavant. Quoique la nature precise de ces granules reste inconnue, il est possible qu'elles soient en relation avec une augmentation du metabolisme des cellules endotheliales, stimulees par l'endocytose et la digestion des particules de ferritine.

Abstract Amphipathic helical peptides represent the lipid-binding units of the soluble plasma apolipoproteins. Several synthetic peptide analogues have been designed to mimic such structures and have been used to unravel some of the mechanisms involved in the physiological function of the apolipoproteins, including lipid binding, LCAT activation, and enhancement of cholesterol efflux from lipid-laden cells. A series of novel synthetic peptides, named ID peptides, was modeled on the basis of the structural properties common to the amphipathic helices of apolipoprotein (apo) A-I. In these new peptides, however, the segregation between hydrophobic and hydrophilic faces of the helices is more pronounced than in apoA-I, so that the surface of the hydrophobic and hydrophilic faces of the amphipathic helices is equal. Moreover, there are fewer negatively charged residues in the center of the hydrophilic face of the helical peptides. Most charged amino acids are located along the edge of the helix and are susceptible to forming salt bridges with residues of an antiparallel helix, such as around a discoidal phospholipid/peptide complex. The physicochemical characteristics of these peptides and their complexes with phospholipids were compared with those of the 18A peptide and its lipid/peptide complex. All ID peptides bind dimyristoylphosphatidylcholine vesicles more rapidly than the 18A peptide to yield discoidal peptide/phospholipid complexes of comparable size. The α-helical content of the lipid-free ID peptides is close to that of the 18A peptide and increases slightly on lipid binding. The stability of the ID and 18A peptides and of the phospholipid/peptide complexes against guanidinium hydrochloride denaturation is higher than that of lipid-free and lipid-bound apoA-I. LCAT activation by the 18A/phospholipid/cholesterol complexes equals that of apoA-I/phospholipid/cholestrol complexes, whereas none of the ID peptides tested is able to activate LCAT to a significant extent. Incubation of the peptide/phospholipid complexes with lipid-laden macrophages induces cellular cholesterol efflux and incorporation of cholesterol into the complexes. The cholesterol efflux capacity of the peptide/phospholipid complexes is comparable among the peptides and higher than that of apoprotein/phospholipid complexes. In conclusion, although the amphipathicity of the new peptides is higher than that of the 18A model peptide, the lack of LCAT activation by the ID peptides suggests that an enhanced segregation of the hydrophobic and hydrophilic residues, equal magnitude of hydrophobic and hydrophilic faces of the helix, and the absence of negatively charged residues in the central part of the hydrophilic face might account for the lack of LCAT activity of these peptides. These parameters do not affect the capacity of the peptide/phospholipid complexes to promote cellular cholesterol efflux.