Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins.

Abstract Background Plasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats. Results In vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p<0.001) but did not modify the transfer of 3H-FC. In hepatectomized rats, heparin stimulation of intravascular lipolysis increased the plasma removal of 14C-PL and the amount of 14C-PL found in the low density lipoprotein density fraction but not in the high density lipoprotein density fraction. The in vitro and in vivo experiments showed that free cholesterol and phospholipids were transferred from lipid emulsion to plasma lipoproteins independently from each other. The incubation of human plasma, control and control plus monoclonal antibody anti-cholesteryl ester transfer protein (CETP), with 14C-PL emulsion showed that CETP increases 14C-PL transfer to human HDL, since its partial inhibition by the anti-CETP antibody reduced significantly the 14C-PL transfer (p<0.05). However, comparing the nontransgenic (no CETP activity) with the CETP transgenic mouse plasma, no effect of CETP on the 14C-PL distribution in mice lipoproteins was observed. Conclusions It is concluded that: 1-intravascular lipases stimulate phospholipid transfer protein mediated phospholipid transfer, but not free cholesterol, from triglyceride rich particles to human high density lipoproteins and rat low density lipoproteins and high density lipoproteins; 2-free cholesterol and phospholipids are transferred from triglyceride rich particles to plasma lipoproteins by distinct mechanisms, and 3 - CETP also contributes to phospholipid transfer activity in human plasma but not in transgenic mice plasma, a species which has high levels of the specific phospholipid transfer protein activity.

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study

Abstract Background Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers. Methods SCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell. Results Size exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution. Conclusions The in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

Characterization of lyophilized liposomes produced with non-purified soy lecithin: a case study of casein hydrolysate microencapsulation

A commercial casein hydrolysate was microencapsulated in liposomes produced with non-purified soy lecithin, cryoprotected with two different disaccharides and lyophilized. The encapsulation efficiency of casein hydrolysate ranged from 30 to 40%. The powders were analyzed by differential scanning calorimetry (DSC), scanning electron micrography (SEM), infrared spectroscopy (FTIR) and wide angle X-ray diffraction (WAXD). DSC data revealed the presence of an exothermal transition in empty lyophilized liposomes, which was ascribed to the presence of a quasicrystalline lamellar phase (intermediary characteristics between the Lβ and Lc phases). The addition of peptides to the liposomal system caused the disappearance of this exothermic phenomenon, as they were located in the polar headgroup portion of the bilayer, causing disorder and preventing the formation of the quasicrystalline phase. Infrared data indicated the presence of the peptides in the lyophilized formulations and showed that the cryoprotectants interacted effectively with the polar heads of phospholipids in the bilayer.

Theoretical Study of the Molecules in Solution and Phospholipid Bilayers

Using Molecular Dynamics simulations and Quantum Mechanical calculations, we study the behavior of molecules with biophysical and pharmacological interest in solution and in phospholipid bilayer.

Mechanics and dynamics of liposomes and cells studied by QCM and ECIS

The present thesis introduces a novel sensitive technique based on TSM resonators that provides quantitative information about the dynamic properties of biological cells and artificial lipid systems. In order to support and complement results obtained by this method supplementary measurements based on ECIS technique were carried out. The first part (chapters 3 and 4) deals with artificial lipid systems. In chapter 3 ECIS measurements were used to monitor the adsorption of giant unilamellar vesicles as well as their thermal fluctuations. From dynamic Monte Carlo Simulations the rate constant of vesicle adsorption was determined. Furthermore, analysis of fluctuation measurements reveals Brownian motion reflecting membrane undulations of the adherent liposomes. In chapter 4 QCM-based fluctuation measurements were applied to quantify nanoscopically small deformations of giant unilamellar vesicles with an external electrical field applied simultaneously. The response of liposomes to an external voltage with shape changes was monitored as a function of cholesterol content and adhesion force. In the second part (chapters 5 - 8) attention was given to cell motility. It was shown for the first time, that QCM can be applied to monitor the dynamics of living adherent cells in real time. QCM turned out to be a highly sensitive tool to detect the vertical motility of adherent cells with a time resolution in the millisecond regime. The response of cells to environmental changes such as temperature or osmotic stress could be quantified. Furthermore, the impact of cytochalasin D (inhibits actin polymerization) and taxol (facilitate polymerization of microtubules) as well as nocodazole (depolymerizes microtubules) on the dynamic properties of cells was scrutinized. Each drug provoked a significant reduction of the monitored cell shape fluctuations as expected from their biochemical potential. However, not only the abolition of fluctuations was observed but also an increase of motility due to integrin-induced transmembrane signals. These signals were activated by peptides containing the RGD sequence, which is known to be an integrin recognition motif. Ultimately, two pancreatic carcinoma cell lines, derived from the same original tumor, but known to possess different metastatic potential were studied. Different dynamic behavior of the two cell lines was observed which was attributed to cell-cell as well as cell-substrate interactions rather than motility. Thus one may envision that it might be possible to characterize the motility of different cell types as a function of many variables by this new highly sensitive technique based on TSM resonators. Finally the origin of the broad cell resonance was investigated. Improvement of the time resolution reveals the "real" frequency of cell shape fluctuations. Several broad resonances around 3-5 Hz, 15-17 Hz and 25-29 Hz were observed and that could unequivocally be assigned to biological activity of living cells. However, the kind of biological process that provokes this synchronized collective and periodic behavior of the cells remains to be elucidated.

Photoactive nanodevices for potential biological applications

Biological systems are complex and highly organized architectures governed by noncovalent interactions, which are responsible for molecular recognition, self-assembly, self-organization, adaptation and evolution processes. These systems provided the inspiration for the development of supramolecular chemistry, that aimed at the design of artificial multicomponent molecular assemblies, namely supramolecular systems, properly designed to perform different operations: each constituting unit performs a single act, whereas the entire supramolecular system is able to execute a more complex function, resulting from the cooperation of the constituting components. Supramolecular chemistry deals with the development of molecular systems able to mimic naturally occurring events, for example complexation and self-assembly through the establishment of noncovalent interactions. Moreover, the application of external stimuli, such as light, allows to perform these operations in a time- and space-controlled manner. These systems can interact with biological systems and, thus, can be applied for bioimaging, therapeutic and drug delivery purposes. In this work the study of biocompatible supramolecular species able to interact with light is presented. The first part deals with the photophysical, photochemical and electrochemical characterization of water-soluble blue emitting triazoloquinolinium and triazolopyridinium salts. Moreover, their interaction with DNA has been explored, in the perspective of developing water-soluble systems for bioimaging applications. In the second part, the effect exerted by the presence of azobenzene-bearing supramolecular species in liposomes, inserted both in the phospholipid bilayer and in the in the aqueous core of vesicles has been studied, in order to develop systems able to deliver small molecules and ions in a photocontrolled manner. Moreover, the versatility of azobenzene and its broad range of applications have been highlighted, since conjugated oligoazobenzene derivatives proved not to be adequate to be inserted in the phospholipid bilayer of liposomes, but their electrochemical properties made them interesting candidates as electron acceptor materials for photovoltaic applications.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of de novo synthesis of all major phospholipids. Here we provide an overview of the biosynthetic pathways involved in phospholipid formation in T. brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis. We show that de novo synthesis of phosphatidylethanolamine involving CDP-activated intermediates is essential in T. brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure. In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggesting a role for phosphatidylcholine formation in the control of cell division. Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T. brucei and review the biosynthesis of phosphatidylinositol, which seems to take place in two separate compartments. Finally, we emphasize that T. brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inositolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated.

Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.

Increased placental phospholipid levels in pre-eclamptic pregnancies

Physiological pregnancy is associated with an increase in lipids from the first to the third trimester. This is a highly regulated response to satisfy energy and membrane demands of the developing fetus. Pregnancy disorders, such as pre-eclampsia, are associated with a dysregulation of lipid metabolism manifesting in increased maternal plasma lipid levels. In fetal placental tissue, only scarce information on the lipid profile is available, and data for gestational diseases are lacking. In the present study, we investigated the placental lipid content in control versus pre-eclamptic samples, with the focus on tissue phospholipid levels and composition. We found an increase in total phospholipid content as well as changes in individual phospholipid classes in pre-eclamptic placental tissues compared to controls. These alterations could be a source of placental pathological changes in pre-eclampsia, such as lipid peroxide insult or dysregulation of lipid transport across the syncytiotrophoblast.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix−hinge−helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.