Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine.

The serum lipoprotein high-density lipoprotein (HDL), which is a ligand of scavenger receptors such as scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36), can act as a donor particle for intestinal lipid uptake into the brush border membrane (BBM). Both cholesterol and phospholipids are taken up by the plasma membrane of BBM vesicles (BBMV) and Caco-2 cells in a facilitated (protein-mediated) process. The protein-mediated transfer of cholesterol from reconstituted HDL to BBMV depends on the lipid composition of the HDL. In the presence of sphingomyelin, the transfer of cholesterol is slowed by a factor of about 3 probably due to complex formation between cholesterol and the sphingolipid. It is shown that the mechanism of lipid transfer from reconstituted HDL to either BBMV or Caco-2 cells as the acceptor is consistent with selective lipid uptake: the lipid donor docks at the membrane-resident scavenger receptors which mediate the transfer of lipids between donor and acceptor. Selective lipid uptake implies that lipid, but no apoprotein is transferred from the donor to the BBM, thus excluding endocytotic processes. The two BBM models used here clearly indicate that fusion of donor particles with the BBM can be ruled out as a major mechanism contributing to intestinal lipid uptake. Here we demonstrate that CD36, another member of the family of scavenger receptors, is present in rabbit and human BBM vesicles. This receptor mediates the uptake of free cholesterol, but not of esterified cholesterol, the uptake of which is mediated exclusively by SR-BI. More than one scavenger receptor appears to be involved in the uptake of free cholesterol with SR-BI contributing about 25% and CD36 about 35%. There is another yet unidentified protein accounting for the remaining 30 to 40%.

Liposomes as protective agents of stratum corneum against octyl glucoside: a study based on high-resolution, low-temperature scanning electron microscopy.

The ability of phosphatidylcholine (PC) liposomes to protect pig stratum corneum (SC) against the action of the nonionic surfactant octyl glucoside (OG) was investigated "in vitro" using double-layer coating for high-resolution, low-temperature scanning electron microscopy. This technique has been useful in preventing drying artifacts in the study of biological materials. The treatment of SC with OG led to a perturbation mainly in the corneocytes. However, the incubation of the tissue with liposomes prior to the OG treatment resulted in a progressive decrease in these perturbations and, consequently, in the progressive protection of the SC against the action of the surfactant.

The major protein import receptor of plastids is essential for chloroplast biogenesis.

Light triggers the developmental programme in plants that leads to the production of photosynthetically active chloroplasts from non-photosynthetic proplastids. During this chloroplast biogenesis, the photosynthetic apparatus is rapidly assembled, mostly from nuclear-encoded imported proteins, which are synthesized in the cytosol as precursors with cleavable amino-terminal targeting sequences called transit sequences. Protein translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes recognize these transit sequences, leading to the precursors being imported. The Toc complex in the pea consists of three major components, Toc75, Toc34 and Toc159 (formerly termed Toc86). Toc159, which is an integral membrane GTPase, functions as a transit-sequence receptor. Here we show that Arabidopsis thaliana Toc159 (atToc159) is essential for the biogenesis of chloroplasts. In an Arabidopsis mutant (ppi2) that lacks atToc159, photosynthetic proteins that are normally abundant are transcriptionally repressed, and are found in much smaller amounts in the plastids, although ppi2 does not affect either the expression or the import of less abundant non-photosynthetic plastid proteins. These findings indicate that atToc159 is required for the quantitative import of photosynthetic proteins. Two proteins that are related to atToc159 (atToc120 and atToc132) probably help to maintain basal protein import in ppi2, and so constitute components of alternative, atToc159-independent import pathways.

Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

Solubilization of liposomes by sodium dodecyl sulfate: new mechanism based on the direct formation of mixed micelles.

The vesicle-to-micelle structural transitions that occurred in the interaction of sodium dodecyl sulfate with phosphatidylcholine vesicles were studied at the equilibrium by means of dynamic light scattering (at different scattering angles) and freeze-fracture electron microscopy techniques. The incorporation of surfactant monomers in the bilayers resulted in an initial contraction of the mixed vesicles formed up to their saturation (size reduction of about 10%). Then, a progressive relaxation of these structures (growth from 170 to 225 nm) and a simultaneous formation of mixed micelles (particles of about 6 nm) occurred. Hence, in this interval "relaxed mixed vesicles" and mixed micelles coexisted in different proportions without formation of intermediate complex aggregates (bimodal size distribution curves). Freeze-fracture electron microscopy showed a direct formation of mixed micelles within the bilayer and their subsequent separation from the vesicle surface without formation of complex intermediate aggregates. This simple process progressed up to the complete vesicle solubilization.

Direct formation of mixed micelles in the solubilization of phospholipid liposomes by Triton X-100.

The vesicle to micelle transition which results in the interaction of the Triton X-100 surfactant with phosphatidylcholine vesicles was studied by means of dynamic light scattering (at different reading angles) and by freeze-fracture electron microscopy techniques. Vesicle solubilization was produced by the direct formation of mixed micelles without the formation of complex intermediate aggregates. Thus, vesicle to micelle transformation was mainly governed by the progressive formation of mixed micelles within the bilayer. A subsequent separation of these micelles from the liposome surface (vesicle perforation by the formation of surfactant-stabilized holes on the vesicle surface) led to a complete solubilization of liposomes.

Identification of a receptor mediating absorption of dietary cholesterol in the intestine.

Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily.

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Preparation and characterization of polyethyl-2-cyanoacrylate nanocapsules containing antiepileptic drugs.

Biocompatible and biodegradable colloidal drug delivery systems can be obtained by means of in situ polymerization of alkylcyanoacrylate. In particular, nanocapsules of polyethylcyanoacrylate (PECA) were prepared by adding the monomer to an organic phase, consisting of Miglyol 812 and an organic solvent (ethanol, acetone or acetonitrile), and subsequently mixing the organic phase with an aqueous phase containing Pluronic F68 at different concentrations. The possible mechanism of formation and the influence of preparation conditions on the quality of nanocapsule formulations were investigated by freeze-fracture electron microscopy and laser light scattering using both the inverse Laplace transform and the standard cumulant analysis for data fitting. High-quality nanocapsule systems were obtained using an aprotic fully water-miscible organic solvent such as acetone. The presence of ethanol led to the formation of both nanospheres and nanocapsules. The concentrations of nonionic surfactant in the aqueous phase of monomer in the organic phase did not influence the kind of colloidal suspension obtained. The oil simply plays the role of monomer support. The diameter of PECA nanoparticles (nanospheres and nanocapsules) ranged from 100 to 400 nm. Three antiepileptic drugs (Ethosuximide, 5,5-diphenyl hydantoin and carbamazepine) were entrapped in PECA nanocapsules. The loading capacity of PECA nanocapsules, prepared using acetone as organic solvent, varied from 1% to 11% (drug/dried material) as a function of the solubility (affinity) of the different drugs with the oil core. This parameter also influenced the release from PECA nanocapsules, which was slower for drugs with a higher affinity for Miglyol 812. By encapsulating the three antiepileptic drugs in the PECA nanocapsules, it was possible to achieve controlled drug release. The mechanism of drug release from PECA nanocapsules was mainly diffusion from the oil core through the intact polymer barrier.

Isolation, characterization and electron microscopy analysis of a hemidiscoidal phycobilisome type from the cyanobacterium Anabaena sp. PCC 7120.

In this work we present the characterization of a hemidiscoidal phycobilisome type of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The phycobilisome of this organism contains allophycocyanin, phycocyanin and phycoerythrocyanin, similar to the closely related thermophilic cyanobacterium Mastigocladus laminosus. Intact phycobilisomes exhibit an absorption maximum at 619 nm and two fluorescence maxima at 664 nm and 680 nm, corroborating the presence of a complete energy pathyway along the antenna. Upon dissociation, the phycobiliproteins were released from the phycobilisome. One phycoerythrocyanin, one phycocyanin and three allophycocyanin complexes were isolated by ion-exchange chromatography and characterized by absorption and fluorescence spectroscopy and by SDS/PAGE. The amino-terminal sequences of the polypeptides belonging to the phycoerythrocyanin and phycocyanin families were identical with the derived sequences of their corresponding genes. Partial amino-terminal sequences of the polypeptides belonging to the allophycocyanin family are presented here. Our results show that the phycobiliproteins and linker polypeptides from Anabaena sp. PCC 7120 are similar to the phycobilisome components characterized in other cyanobacteria. The phycobilisome of Anabaena sp. PCC 7120 was extensively analyzed by electron microscopy. It differs from the common hemidiscoidal tricylindrical, six-rod phycobilisome type by a core domain consisting of five core cylinders surrounded by up to eight rods radiating in a hemidiscoidal manner. One rod is linked to each basal core cylinder, whereas the remaining core cylinders bind two rods each. On the basis of the data presented in this work, a revised model for the hemidiscoidal pentacylindrical phycobilisome of Anabaena sp. PCC 7120, M. laminosus and Anabaena variabilis is proposed. This model accounts more accurately for the 'grape' pattern typically exhibited by these phycobilisomes in electron micrographs.

[Small-caliber polyurethane arterial prosthesis: clinical and angiomorphological follow-up of 20 patients in a prospective study]. / Prothèses artérielles de petit calibre en polyuréthane: Suivi clinique at angio-morphologique de 20 patients d'une étude prospective.

The five year patency rate for femoropopliteal vein bypass grafts is around 70% according to the literature. Patency rates for synthetic grafts (eg PTFE, Dacron) range between 43 and 57%. If a vein is not available there is a new polyurethane 6 mm artery substitute on the market, that has shown in vitro promising physical characteristics and good long term results after implantation in dogs. In a prospective, randomized trial the results of the new polyurethane graft (PUR) were compared with those of a Dacron graft of the same diameter. Included in the study were 20 patients with lower limb ischemia stage Fontaine II B, III and IV, 10 in each group. Patency rates, handling of the graft and complications were analysed. During the one year follow up 7 PUR grafts had to be changed due to recurrent bypass occlusion within the first 3 months. At the end of the year there were only one PUR-bypass but 8 Dacron grafts open. 5 PUR grafts were examined histologically and no morphological reason for the occlusion, especially no myointimal hyperplasia, was found. A special regard was brought to the arterial run-off in both groups. It was confirmed to be comparable with only slightly better data for the PUR group. The exact reasons for the astonishing bad results of the PUR graft for femoropopliteal above knee bypass cannot be explained in our study. Due to the unexpected high occlusion rate the study was stopped earlier then planned.

Enhanced therapeutic effect of cytidine-5'-diphosphate choline when associated with GM1 containing small liposomes as demonstrated in a rat ischemia model.

PURPOSE: Cytidine-5'-diphosphate choline (CDPc) was encapsulated in long-circulating unilamellar vesicles (SUVs) to improve the drug's biological effectiveness. METHODS: SUVs made up of diaplmitoylphosphatidylcholine/diaplmitoylphosphatidylserine /cholesterol (7:4:7 molar ratio) and 8 mol % of ganglioside GM1 were prepared by extrusion through polycarbonate filters (mean diameter 50 nm). The formulation effectiveness was evaluated by an in vivo model of cerebral ischemia on Wistar rats. RESULTS: The enhanced delivery of CDPc into the brain improved the therapeutic effectiveness of the drug. CDPc-loaded SUVs improved the survival rate of ischemized and reperfused Wistar rats (320-350 g) by approximately 66% compared with the free drug. Liposome formulation was also able to effectively protect the brain against peroxidative damage caused by post-ischemic reperfusion. SUVs lowered the conjugated diene levels of the cerebral cortex. The liposomal delivery system did not alter the distribution patterns in the various cerebral lipid fractions of the drug, radiolabeled with 14C-CDPc. CONCLUSIONS: CDPc-loaded SUVs were able to protect the brain against damage induced by ischemia. A possible clinical application is envisaged.

Double-layer coating for high-resolution low-temperature scanning electron microscopy.

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in low-temperature scanning electron microscopy of bulk specimens. At high primary magnifications (e.g., 100,000 x) a hydrated sample is usually severely damaged after one slow scan (about 3000 e-nm-2). The consequences of this beam damage are significantly reduced by coating the frozen-hydrated sample with a 5-10-nm-thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g., 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field-emission SEM and a sensitive backscattered electron detector, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.

Neutrase entrapment in stable multilamellar and large unilamellar vesicles for the acceleration of cheese ripening.

We report the encapsulation of neutrase in liposomes for the acceleration of cheese ripening. The liposome preparation procedure consisted of repeated freeze-thaw cycles of multilamellar vesicles followed by extrusion through polycarbonate filters. The neutrase encapsulation efficiency in the liposomes was influenced by the number of freeze-thaw cycles, achieving the highest value after seven cycles. Filtration through 200-nm polycarbonate membranes yielded homogenous size liposome populations with trapping efficiencies of about 65%. The vesicle stability and low neutrase release during the first stages of the cheese-making procedure, coupled with an almost quantitative retention of neutrase-loaded liposomes in cheese curd, ensured a proteolysis rate that was twice that observed in the control cheese.

Characterization of lipid exchange proteins isolated from small intestinal brush border membrane.

Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 +/- 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an integral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.

Natural language processing, lexicon and semantics.

While the design of a fully general procedure for semantico-pragmatic interpretation of natural language texts does not seem to be feasible with current scientific knowledge and technology, the more practical microworld based approaches lack generality and portability. A compromise between generality and practicality might lie in the use of an intermediate level of presentation ("pseudo-semantics"), which can be derived from syntactic representations and lexical information by means of a general procedure. Domain-dependent rules for semantico-pragmatic interpretation can then be applied to these representations, insulating syntactic processing from details of the application domain.

Quality improvement of spray-dried, protein-loaded D,L-PLA microspheres by appropriate polymer solvent selection.

The aim was to study the effect of the type of polymer solvent on characteristics of microspheres produced by spray drying. The water-soluble model protein, bovine serum albumin (BSA) was microencapsulated into biodegradable poly(D,L-lactic acid) using the following 10 different polymer solvents: acetaldehyde dimethyl acetal, acetone, dichloromethane, dioxane, ethyl acetate, ethyl vinyl ether, nitromethane, tetrahydrofuran, 1,1,1-trichloroethane, and 1,1,2-trichloroethylene. These solvents having similar toxicity levels differ greatly in their physico-chemical characteristics such as boiling point, vapour pressure, miscibility and interfacial tension with an aqueous phase, and solubility parameter. The effect of these solvents on microsphere morphology was studied by SEM-micrographs. Regular particle morphology was obtained when dichloromethane, ethyl acetate, or nitromethane was used as the polymer solvent, whereas the trichlorinated solvents, tetrahydrofuran, and dioxane produced a substantial number of coalesced particles. The results are interpreted in terms of boiling point, vapour pressure, and polymer-solvent affinity. Further, BSA-loading and -integrity in the microspheres, and burst release were analysed. The theoretical loading of 2.9% was attained with dichloromethane, ethyl acetate and nitromethane, in agreement with observations of particle morphology. HPLC- and SDS-PAGE analysis of the microencapsulated BSA did not show any protein degradation or dimerization, whereas solid-phase ELISA clearly revealed that the in vitro protein antigenicity was substantially reduced (50%), particularly by water miscible solvents. Dichloromethane and ethyl acetate did not show any detrimental effect on protein antigenicity. Finally, burst release could be related again to particle morphology, with dichloromethane and nitromethane giving a burst release of only 5%. In conclusion, dichloromethane, ethyl acetate and nitromethane proved to be the most suitable solvents for the polymer-protein system studied.

Short-chain phosphatidylcholines as superior detergents in solubilizing membrane proteins and preserving biological activity.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%). The size of the resulting mixed protein-lipid-DHPC micelles ranges between 5 and 8 nm. The protein conformation and hence the enzymatic activity are well preserved over a rather large DHPC concentration range (up to 4-5 times the DHPC concentration required for solubilizing the membranes). Evidence is presented that short-chain phosphatidylcholines are superior to most detergents commonly used by biochemists. This is true not only regarding its excellent dispersing power on both phospholipid bilayers (Gabriel & Roberts, 1986) and biological membranes but also as to its capacity to preserve the native protein structure and hence enzymatic activity in the solubilized state. Due to its special properties DHPC lends itself very well not only to membrane solubilization but also to the purification of the solubilized membrane proteins and reconstitution of the proteins into simple lipid bilayers. Concerning the mechanism of membrane solubilization, evidence indicates that DHPC interacts primarily with the lipid bilayer of the membrane and not with the membrane proteins. DHPC solubilizes membranes by being distributed into the lipid bilayer and breaking it up. In the resulting small mixed micelles, the protein remains associated with its preferred intrinsic membrane lipids and is thus stabilized. The protein-intrinsic lipid complex is successfully shielded from unfavorable contacts with H2O by DHPC-intrinsic lipid interactions.

Thermodynamic stability and osmotic sensitivity of small unilamellar phosphatidylcholine vesicles.

Evidence is presented to show that small unilamellar phosphatidylcholine vesicles with a diameter of approx. 20 nm are osmotically sensitive. Such vesicles respond to osmotic pressure by swelling or shrinking depending on the direction of the applied salt gradient. This is true for small unilamellar vesicles of egg phosphatidylcholine and dimyristoylphosphatidylcholine below and above their crystal-to-liquid crystal transition temperature. At the transition temperature the vesicles are osmotically insensitive due to the increased bilayer permeability resulting in rapid dissipation of salt gradients. Positive salt gradients produce shrinking and collapse of spherical phospholipid vesicles to disks. Shrinking of vesicles is associated with H2O and solute efflux, but only limited solute influx. Clustering of lipid molecules in the bilayers of the resulting disks can be detected by EPR spin labeling. Negative salt gradients produce swelling of vesicles which is associated with H2O and solute influx. Our experiments are consistent with an osmotically perturbed bilayer. In the presence of osmotic gradients the influx and efflux of H2O is coupled with the movement of ions and small molecules which in the absence of salt gradients or osmotic stress cannot pass the phospholipid bilayer. However, during osmotically induced shrinking and swelling of SUV the integrity of the phospholipid bilayer is maintained to the extent that vesicles do not break, and therefore equilibration between external medium and vesicle cavity does not take place.