Amália S. Jurado

Mitochondrial membrane lipid remodeling in pathophysiology: a new target for diet and therapeutic interventions.

Mitochondria are arbiters in the fragile balance between cell life and death. These organelles present an intricate membrane system, with a peculiar lipid composition and displaying transverse as well as lateral asymmetry. Some lipids are synthesized inside mitochondria, while others have to be imported or acquired in the form of precursors. Here, we review different processes, including external interventions (e.g., diet) and a range of biological events (apoptosis, disease and aging), which may result in alterations of mitochondrial membrane lipid content. Cardiolipin, the mitochondria lipid trademark, whose biosynthetic pathway is highly regulated, will deserve special attention in this review. The modulation of mitochondrial membrane lipid composition, especially by diet, as a therapeutic strategy for the treatment of some pathologies will be also addressed.

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: A biophysical approach to transfection efficiency

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C(14)H(29)(CH(3))(2)N(+)(CH(2))(2)N(+)(CH(3))(2)C(14)H(29)]2Br(-) and herein designated 14-2-14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14-2-14, 14-2-14:Chol (1:1 molar ratio) and 14-2-14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/- charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14-2-14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/- charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.

Physical studies on membrane lipids of Bacillus stearothermophilus temperature and calcium effects

Bacillus stearothermophilus was grown at the optimal temperature range (center, 65 degrees C), below it (48 and 55 degrees C), and above it (68 degrees C), in a complex medium with or without 2.5 mM Ca2+. The Ca(2+)-supplement improves growth at sub- and supraoptimal temperatures and extends it to higher temperatures (Jurado et al. (1987) J. Gen. Microbiol. 133, 507-513). The phospholipid composition of cultures obtained in the different growth conditions was studied. Phosphatidylethanolamine was always the major phospholipid (40 to 50% of the total phospholipid). Diphosphatidylglycerol, phosphatidylglycerol, a phosphoglycolipid (pgl) and two minor phospholipids (not identified) were also found in the polar lipid extract. The pgl shows a threefold concentration increase as the growth temperature raises from 48 to 68 degrees C. The thermotropic behavior of membrane lipids was studied by differential scanning calorimetry (DSC) and by means of two fluorescent probes of fluidity, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(2-pyrenyl)propane (2Py(3)2Py). The results reveal similar features and clearly show a shift of the temperature range of the phase transition to higher values and an increased structural order of the bilayer, as the growth temperature rises from 55 to 68 degrees C, but an opposite effect was observed from 48 to 55 degrees C. Although the Ca(2+)-supplement to the growth medium has no detectable effect, the addition of Ca2+ to the buffer of liposomes (Ca(2+)-liposomes) has a significant ordering effect at all growth temperatures. These liposomes show a shift of the transition range to higher temperatures and the fluorescent parameters (DPH polarization and intramolecular excimerization of the 2Py(3)2Py) detected an order increase of the probes environment, along and above the main phase transition. Spectra of 31P-NMR and polarized light microscopy clearly show that the lipid extracts exhibit, in all the conditions, typical lamellar phase geometry. We concluded that B. stearothermophilus controls the membrane lipid composition to compensate for the destabilizing effect of high temperatures on the membrane organization or to provide an appropriate packing of phospholipid molecules in a stable bilayer. At high temperatures, Ca(2+)-stimulatory effect on growth is presumably due to a direct Ca2+ interaction with the membrane phospholipids, inducing an increased structural order on the bilayer. The increase of the phase transition temperature in the total lipid extracts as compared with the respective polar lipid fractions probably indicates a stabilizing effect of neutral lipids on membrane bilayers.

Interaction of Fullerene Nanoparticles With Biomembranes: From the Partition in Lipid Membranes to Effects on Mitochondrial Bioenergetics

Partition and localization of C60 and its derivative C60(OH)18-22 in lipid membranes and their impact on mitochondrial activity were studied, attempting to correlate those events with fullerene characteristics (size, surface chemistry, and surface charge). Fluorescence quenching studies suggested that C60(OH)18-22 preferentially populated the outer regions of the bilayer, whereas C60 preferred to localize in deeper regions of the bilayer. Partition coefficient values indicated that C60 exhibited higher affinity for dipalmitoylphosphatidylcholine and mitochondrial membranes than C60(OH)18-22. Both fullerenes affected the mitochondrial function, but the inhibitory effects promoted by C60 were more pronounced than those induced by C60(OH)18-22 (up to 20 nmol/mg of mitochondrial protein). State 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrate. Phosphorylation system and electron transport chain of mitochondria are affected by both fullerenes, but only C60 increased the inner mitochondrial membrane permeability to protons, suggesting perturbations in the structure and dynamics of that membrane. At concentrations of C60(OH)18-22 above 20 nmol/mg of mitochondrial protein, the activity of FoF1-ATP synthase was also decreased. The evaluation of transmembrane potential showed that the mitochondria phosphorylation cycle decreased upon adenosine diphosphate addition with increasing fullerenes concentration and the time of the repolarization phase increased as a function of C60(OH)18-22 concentration. Our results suggest that the balance between hydrophilicity and hydrophobicity resulting from the surface chemistry of fullerene nanoparticles, rather than the cluster size or the surface charge acquired by fullerenes in water, influences their membrane interactions and consequently their effects on mitochondrial bioenergetics.

The effect of cationic gemini surfactants upon lipid membranes. An experimental and molecular dynamics simulation study

Gemini surfactants possess interesting interfacial and aggregation properties that have prompted comprehensive studies and successful applications in a wide variety of fields. However, a systematic study on the effect of gemini tail and spacer length upon the organization of lipid membranes has not been presented so far. In this study, we analyze the action of dicationic alkylammonium bromide gemini surfactants on DPPC liposomes, the latter employed as a model of lipid membranes. Differential scanning calorimetry results indicate that the surfactants presenting shorter tails (12 carbons) induce a decrease in the overall order of the bilayer, while those with longer tails (16 and 18 carbons) lead to the formation of more ordered structures. The respective influence on the degree of lipid order transverse to the bilayer was additionally studied resorting to a detailed fluorescence anisotropy study. In this case, it is observed that among the shorter tail surfactants, those with longer spacers (6 and 10 carbons) are responsible for a more pronounced disrupting effect upon the membrane, especially close to the lipid polar heads. Molecular dynamics simulation supports the most important findings and provides insight into the mechanism that governs this interaction. Accordingly, the interplay between tail and spacer length accounts for the differential vertical positioning of the gemini molecules and atom-density in the core of the bilayer, that provide a rationale for the experimental observations.

Lipid composition and dynamics of cell membranes of Bacillus stearothermophilus adapted to amiodarone

Bacillus stearothermophilus, a useful model to evaluate membrane interactions of lipophilic drugs, adapts to the presence of amiodarone in the growth medium. Drug concentrations in the range of 1-2 microM depress growth and 3 microM completely suppresses growth. Adaptation to the presence of amiodarone is reflected in lipid composition changes either in the phospholipid classes or in the acyl chain moieties. Significant changes are observed at 2 microM and expressed by a decrease of phosphatidylethanolamine (relative decrease of 23.3%) and phosphatidylglycerol (17.9%) and by the increase of phosphoglycolipid (162%). The changes in phospholipid acyl chains are expressed by a decrease of straight-chain saturated fatty acids (relative decrease of 12.2%) and anteiso-acids (22%) with a parallel increase of the iso-acids (9.8%). Consequently, the ratio straight-chain/branched iso-chain fatty acids decreases from 0. 38 (control cultures) to 0.30 (cultures adapted to 2 microM amiodarone). The physical consequences of the lipid composition changes induced by the drug were studied by fluorescence polarization of diphenylhexatriene and diphenylhexatriene-propionic acid, and by differential scanning calorimetry. The thermotropic profiles of polar lipid dispersions of amiodarone-adapted cells are more similar to control cultures (without amiodarone) than those resulting from a direct interaction of the drug with lipids, i.e., when amiodarone was added directly to liposome suspensions. It is suggested that lipid composition changes promoted by amiodarone occur as adaptations to drug tolerance, providing the membrane with physico-chemical properties compatible with membrane function, counteracting the effects of the drug.

Influence of Divalent Cations on the Growth and Morphology of Bacillus stearothermophilus

SUMMARY: Bacillus stearothermophilus grows within the temperature range 40–70°C in a complex medium that contains 115 μM-Ca2+ and 95 μm-Mg2+. The addition of Ca2+ to a final concentration ranging from 2·5 to 10 mm stimulated growth at suboptimal and supraoptimal temperatures, extending growth above 70°C, but had no effect on growth within the optimal temperature range. Mg2+ (2·5 mm) also stimulated growth although to a lesser extent. Furthermore, 10 mm-Mg2+ inhibited growth at temperatures higher than 65°C. This inhibitory effect was relieved by the addition of 2·5 mm-Ca2+. Sr2+ (10 mm), which often behaves as a Ca2+ analogue in biological systems, strongly inhibited growth and produced gross morphological alterations in the cells. The inhibitory effect of Sr2+ could also be relieved by addition of Ca2+.

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

LIPID COMPOSITION CHANGES INDUCED BY TAMOXIFEN IN A BACTERIAL MODEL SYSTEM

A putative relationship between growth impairment of Bacillus stearothermophilus by tamoxifen (TAM) and TAM-induced perturbation of the physical properties of bacterial membrane lipids has been observed. The supplementation of the growth medium with Ca2+ (a membrane stabilizer) partially relieves growth inhibition by TAM, allowing growth at TAM concentrations that fully impair growth in the basal medium. B. stearothermophilus modifies the membrane lipid composition in response to the addition of TAM to the growth medium and the response is sensitive to Ca2+. Changes in lipid composition are observed in the acyl chains and in the polar head groups of phospholipids. The physical effects of alteration in these lipids was studied by fluorescence polarization of DPH and DPH-PA. Polar lipid dispersions from TAM-adapted cells grown in a Ca2+ medium show a shift of Tm to higher temperatures and a significant increase of the structural order as compared to lipids from control cells, suggesting that TAM-induced lipid composition changes compensate for the destabilizing effects of the cytostatic on membrane organization. The polar lipids from cells grown in the basal medium containing tamoxifen are also altered, but these alterations do not promote order increase of the bilayer in spite of a deviation of Tm to higher temperatures as detected by DPH. Data indicate that B. stearothermophilus controls the membrane lipid composition in response to tamoxifen, to compensate for TAM-promoted disordering in membranes and to provide an appropriate packing of phospholipid molecules in a stable bilayer, putatively disturbed by TAM incorporation.