PDMS-based porous particles as support beds for cell immobilization: bacterial biofilm formation as a function of porosity and polymer composition.

The objective of this work is to test the performance of new synthetic polydimethylsiloxane (PDMS)-based bed particles acting as carriers for bacteria biofilms. The particles obtained have a highly interconnected porous structure which offers a large surface adsorption area to the bacteria. In addition, PDMS materials can be cross-linked by copolymerization with other polymers. In the present work we have chosen two hydrophilic polymers: xanthan gum polysaccharide and tetraethoxysilane (TEOS). This versatile composition helps to modulate the interfacial hydrophobic/hydrophilic balance at the particle surface level and the roughness topology and pore size distribution, as revealed by scanning electron microscopy. Biofilm formation of a consortium isolated from a tannery effluent enriched in Sulphate Reducing Bacteria (SRB), and pure Acidithiobacillus ferrooxidans (AF) strains were assayed in three different bed particles synthesized with pure PDMS, PDMS-xanthan gum and PDMS-TEOS hybrids. Bacterial viability assays using confocal laser scanning fluorescence microscopy indicate that inclusion of hydrophilic groups on particle's surface significantly improves both cell adhesion and viability.

A stability test of liposome preparations using steady-state fluorescent measurements.

The stability of liposome preparations under the action of the nonionic detergent Triton X-100 was measured using the fluorescent molecular probe octadecylrhodamine B (R18). The probe inserted in the lipid bilayer shows a self-quenched fluorescence and the degree of quenching depends both on the probe concentration and the phase state of the lipid membrane. The addition of detergent to the liposomes produces a steep decrease in self-quenching caused by dilution of the probe in the bilayer. The curves of steady-state fluorescence intensity show an abrupt change in slope that corresponds to the point at which liposomes break down into lipid-detergent mixed entities that are different from the earlier liposome-monodisperse population. The lytic process was followed in parallel by dynamic light scattering (DLS), and the analysis of the DLS results agree with the interpretation of the fluorescence measurements. The probe R18 therefore is a useful marker to test the stability of liposome preparations. The advantages of the present method are discussed by comparison with other techniques.

Lipid-protein interactions in rat renal subcellular membranes: a biophysical and biochemical study.

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.

Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures.

Lactobacillus acidophilus CRL 640 grown at 25 and 37 degrees C showed a high content of cardiolipin, phosphatidylglycerol, and glycolipids. Cultures grown at 25 degrees C showed a twofold increase in glycolipids in relation to phospholipids, a twofold increase in the C16:0 and a fourfold increase in the C18:2 fatty acids. In contrast, the C19-cyc and the 10-hydroxy acid (C18:0-10 OH) species showed a noticeable decrease. Extracts of total lipids of bacteria grown at 25 and 37 degrees C dispersed in water yielded particles having a high negative surface potential as measured by electrophoretic mobility. Vesicles prepared by extrusion of these dispersions through polycarbonate membranes of 100-nm pore diameter showed high trapping of carboxyfluorescein (CF), which remained unchanged for at least 20 h. The fluorescence anisotropy measured with diphenylhexatriene (DPH) and the generalized polarization of Laurdan were significantly lower in vesicles prepared with lipids containing the highest glycolipid ratio, in comparison to those of bacteria grown at 37 degrees C. No phase transition was detected between 5 and 50 degrees C as measured with both probes. In accordance with these results, no significant release of the trapped CF in this range of temperature was detected. Bile salts and NaCl promoted an increase in the fluorescence, which is interpreted as a change in the permeability properties of the membrane. This effect was lower with KCl, while CaCl2 did not cause any change. The greater permeability change was observed in vesicles with a low glycolipid/phospholipid ratio. NaCl did not affect the packing of the interface as measured with Laurdan, in contrast to CaCl2. The action of Ca+2 may be ascribed to the binding to the negatively charged lipids, such as phosphatidyl glycerol and cardiolipin. It is concluded that the higher glycolipid/phospholipid ratio and the fatty acids C18:2 and C16:0 enhance the lipid membrane stability and decrease the organization in the interfacial and hydrocarbon zones. These results are congruent with the behavior of entire bacteria subject to osmotic and freeze/thaw stresses.

Phase and surface properties of lipid bilayers containing neoglycolipids.

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25 degrees C.

Gel state surface properties of phosphatidylcholine liposomes as measured with merocyanine 540.

The surface properties of liposomes composed by saturated phosphatidylcholines and their mixtures with cholesterol in the gel state have been studied using merocyanine 540 as a fluorescent and optical probe. A new absorption peak at 450 nm and a new fluorescent band at 630 nm were observed when the dye was added to suspensions of DMPC multilamellar liposomes in the gel state. These peaks were also observed in membranes with different lipid compositions in conditions in which the P beta' and the L beta' phases were present. The increase of temperature above the main transition temperature of DMPC or the incorporation of 35% cholesterol into DMPC bilayers at 13 degrees C caused the disappearance of these peaks. The changes in the absorption and fluorescent spectra upon addition of cholesterol resembles very well the phase diagrams reported by Mortensen et al. ((1988) Biochim. Biophys. Acta 945, 221-245) indicating that the corrugated structures characteristic of the L beta' and the P beta' phases have different surface properties related to the partitioning of amphiphilic dies.

Modulatory effects of phosphatidylserine on the binding of muscarinic cholinergic receptor ligands. Studies in vitro and in vivo.

The modulation of the binding of muscarinic cholinergic receptor ligands by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was examined in vitro and in vivo. The enrichment of bovine cerebral cortical synaptosomal membranes with BC-PS, using a fusion technique, produced a concentration-dependent decrease in the affinity (increase in Kd) of [3H]quinuclidinyl benzylate (3H-QNB) specific binding to muscarinic acetylcholine receptors (mAChR), without changes in their maximal number (Bmax). Similar results were observed when [3H]oxotremorine (3H-OXO) was used to label a high affinity subpopulation of mAChR. On the other hand, preincubation of BC-PS liposomes with synaptosomal membranes in a nonoptimum fusion condition (at pH 7.4) did not alter the binding properties of both radioligands. Fusion experiments using a pure phosphatidylserine preparation from spinal cord revealed a similar decrement in the affinity of 3H-QNB specific binding. Five day's intraperitoneal (i.p.) administration of 15 mg/kg of BC-PS liposomes in rats increased the maximal number of cerebral cortical binding sites for 3H-OXO. Scatchard analysis revealed no changes in the apparent dissociation constant. This modification is selective in relation to the neural structure studied. Thus, BC-PS treatment did not modify 3H-OXO binding in the hippocampal formation and cerebellum. In contrast, parallel experiments using the muscarinic antagonist 3H-QNB showed no alteration in the binding properties of mAChR. Five day's i.p. administration of 15 mg/kg/d of phosphatidylcholine from bovine cerebral cortex (BC-PC) liposomes produced quite similar results to those obtained with BC-PS. These results indicate that mAChR are under the modulatory action of phosphatidylserine (PS) and phosphatidylcholine (PC), and suggest that this endogenous phospholipids may play a regulatory role on the mAChR. The possible implications of these findings on the effects of PC or PS treatment in neurological disorders involving a decrease in central cholinergic functions are discussed.