Activation of phospholipase A2 by prostaglandin in vitro.

Prostaglandins are a diverse family of biological active molecules that are synthesized after liberation of arachnidonic or linolenic acid from the plasma membrane by phospholipase A2 (PLA2). Specific prostaglandins may be pro-inflammatory or anti-inflammatory due to a poorly understood biochemical equilibrium. Some of the anti-inflammatory prostaglandins namely, prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) have a cyclopentenone moiety that can react and modify a protein's activity. These two prostaglandins are electrophilic reactive lipid species and are formed as a result of the reaction cascade initiated by PLA2. It was of interest to study the interaction with these prostaglandins as they could either amplify or block this enzyme's activity. We found that the former is true initially as there is a shorter time to activate the protein on the lipid membrane and an overall increase in hydrolysis was observed when PGA1 and PGE1 prostaglandin was added with PLA2 and liposomes. The interfacial activation model was further explored in which there is a modification of the enzyme rather than a modifcation of the substrate. However, after a time the protein was shown to form amyloid like fibrils thereby blocking further hydrolysis. The fibrillization kinetics in the presence of the one of the prostaglandins was monitored using thioflavin T (ThT) and the resulting fibrils were characterized using transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modification of PLA2 by these prostaglandins leading to amyloid like fibrils gives an additional perspective of control of the interfacial activation mechanism of this enzyme.

Interactions and dynamics of two extended conformation adapting phosphatidylcholines in model biomembranes.

In order to obtain molecular level insight into the biophysics of the apoptosis promoting phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) we studied its partitioning into different lipid phases by isothermal titration calorimetry (ITC). To aid the interpretation of these data for PazePC, we additionally characterized by both ITC and fluorescence spectroscopy the fluorescent phospholipid analog 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-C6-PC), which similarly to PazePC can adopt extended conformation in lipid bilayers. With the NBD-hexanoyl chain reversing its direction and extending into the aqueous space out of the bilayer, 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) becomes accessible to the water soluble dithionite, which reduces to non-fluorescent product. Our results suggest that these phospholipid derivatives first partition and penetrate into the outer bilayer leaflet of liquid disordered phase liposomes composed of unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Upon increase up to 2 mol% PazePC and NBD-C6-PC of the overall content, flip-flop from the outer into the inner bilayer leaflet commences. Interestingly, the presence of 40 mol% cholesterol in POPC liposomes did not abrogate the partitioning of PazePC into the liquid ordered phase. In contrast, only insignificant partitioning of PazePC and NBD-C6-PC into sphingomyelin/cholesterol liposomes was evident, highlighting a specific membrane permeability barrier function of this particular lipid composition against oxidatively truncated PazePC, thus emphasizing the importance of detailed characterization of the biophysical properties of membranes found in different cellular organelles, in terms of providing barriers for lipid-mediated cellular signals in processes such as apoptosis. Our data suggest NBD-C6-PC to represent useful fluorescent probe to study the cellular dynamics of oxidized phospholipid species, such as PazePC.

Novel endosomolytic peptides for enhancing gene delivery in nanoparticles.

Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.

Phospholipid lateral diffusion in phosphatidylcholine-sphingomyelin-cholesterol monolayers; effects of oxidatively truncated phosphatidylcholines.

Oxidative stress is involved in a number of pathological conditions and the generated oxidatively modified lipids influence membrane properties and functions, including lipid-protein interactions and cellular signaling. Brewster angle microscopy demonstrated oxidatively truncated phosphatidylcholines to promote phase separation in monolayers of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). More specifically, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), was found to increase the miscibility transition pressure of the SM/Chol-phase. Lateral diffusion of lipids is influenced by a variety of membrane properties, thus making it a sensitive parameter to observe the coexistence of different lipid phases, for instance. The dependence on lipid lateral packing of the lateral diffusion of fluorophore-containing phospholipid analogs was investigated in Langmuir monolayers composed of POPC, SM, and Chol and additionally containing oxidatively truncated phosphatidylcholines, using fluorescence correlation spectroscopy (FCS). To our knowledge, these are the first FCS results on miscibility transition in ternary lipid monolayers, confirming previous results obtained using Brewster angle microscopy on such lipid monolayers. Wide-field fluorescence microscopy was additionally employed to verify the transition, i.e. the loss and reformation of SM/Chol domains.

Acyl Chain Disorder and Azelaoyl Orientation in Lipid Membranes Containing Oxidized Lipids.

Oxidized phospholipids occur naturally in conditions of oxidative stress and have been suggested to play an important role in a number of pathological conditions due to their effects on a lipid membrane acyl chain orientation, ordering, and permeability. Here we investigate the effect of the oxidized phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) on a model membrane of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using a combination of (13)C-(1)H dipolar-recoupling nuclear magnetic resonance (NMR) experiments and united-atom molecular dynamics (MD) simulations. The obtained experimental order parameter SCH profiles show that the presence of 30 mol % PazePC in the bilayer significantly increases the gauche content of the POPC acyl chains, therefore decreasing the thickness of the bilayer, although with no stable bilayer pore formation. The MD simulations reproduce the disordering effect and indicate that the orientation of the azelaoyl chain is highly dependent on its protonation state with acyl chain reversal for fully deprotonated states and a parallel orientation along the interfacial plane for fully protonated states, deprotonated and protonated azelaoyl chains having negative and positive SCH profiles, respectively. Only fully or nearly fully protonated azelaoyl chain are observed in the (13)C-(1)H dipolar-recoupling NMR experiments. The experiments show positive SCH values for the azelaoyl segments confirming for the first time that oxidized chains with polar termini adopt a parallel orientation to the bilayer plane as predicted in MD simulations.

Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology.

Heat shock protein 70 (Hsp70) is an evolutionarily highly conserved molecular chaperone that promotes the survival of stressed cells by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced cell death. Clues to its molecular mechanism of action may lay in the recently reported stress- and cancer-associated translocation of a small portion of Hsp70 to the lysosomal compartment. Here we show that Hsp70 stabilizes lysosomes by binding to an endolysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingomyelin metabolism. In acidic environments Hsp70 binds with high affinity and specificity to BMP, thereby facilitating the BMP binding and activity of acid sphingomyelinase (ASM). The inhibition of the Hsp70-BMP interaction by BMP antibodies or a point mutation in Hsp70 (Trp90Phe), as well as the pharmacological and genetic inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM-is also associated with a marked decrease in lysosomal stability, and this phenotype can be effectively corrected by treatment with recombinant Hsp70. Taken together, these data open exciting possibilities for the development of new treatments for lysosomal storage disorders and cancer with compounds that enter the lysosomal lumen by the endocytic delivery pathway.

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein.

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-∆SBD and Hsp70-∆NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD. Association of Hsp70 with phosphatidylcholine (PC) liposomes is weak, with insertion of its Trps into the bilayer hydrocarbon region. In the presence of cardiolipin (CL), bis-monoacylglycero phosphate (BMP), or phosphatidylserine (PS) Hsp70 attaches to membranes peripherally, without penetration. Our data suggest that the organelle distribution of Hsp70 is determined by their specific lipid compositions, with Hsp70 associating with the above lipids in mitochondria, lysosomes, and the surface of cancer cells, respectively. NBD and SBD attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with Hsp70 in the extended conformation with acyl chains inserting into hydrophobic crevices within Hsp70, and other chains remaining in the bilayer. This anchorage is expected to cause a stringent orientation of Hsp70 on the surface. Our data further suggest that acidic phospholipids induce a transition of SBD into the molten globule state, which may be essential to allow SBD-substrate interaction also within the hydrophobic bilayer interior acyl chain region.

Correction: Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes.

Correction for 'Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes' by Paavo K. J. Kinnunen et al., Soft Matter, 2015, DOI: 10.1039/b925228b.

Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes.

The conversion of various and to is accelerated by , which are also postulated to represent targets mediating the cytotoxicity of protofibrils. Yet, our understanding of the molecular details governing -catalyzed fibrillogenesis of precursors remains limited. To obtain insight into the intricate interplay of and biophysics we have recently introduced supported bilayers (SLBs) with fluorescent analogs as model biomembranes, observed by time-lapse . Here we demonstrate that human islet () induces within minutes of its application on bilayers the expulsion of numerous flexible tubules from the . Intriguingly, these flexible tubules gradually evolve into a network of straight tubes locally attached to the substrate. Two-color imaging of the and the fluorescently labeled revealed to be distributed along the . Similar linear tubules were observed with the antimicrobial temporin B and the non-amyloidogenic rat , revealing that the above mesoscopic perturbations are not related to formation by the human . Micromanipulation experiments revealed that the linearity of the tubules was caused by tension, stretching the tubules between their points of attachment to the substrate. After longer incubation times, for SLBs containing the oxidatively modified 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (, bearing a terminal carboxyl at the end of the chain) and human (but not the other ) some of the straight transformed into highly regular helices. This is likely to reflect tension originating from an efficient aggregation of the into parallelly aligned bundles, associated with tubes containing the oxidized , possibly together with a concomitant flow of along the tubules to the immobile aggregates attaching the tubules to the substrate, these two processes cause, upon shortening of the linear scaffold, the attached excess tubule to adopt a helical morphology, coiling around the core. The above studies are in line with the multiphasic kinetics of fibrillation in the presence of oxidized containing liposomes, assessed by fluorescence enhancement. In addition to demonstrating the feasibility of SLBs as biomimetic model system for studying -assisted fibrillation, our results accentuate the role of chemical composition in modulation of different stages of this process and the associated transformation of architecture. Accordingly, changes in the chemical nature of cellular arising from pathophysiological processes such as oxidative stress may participate in the triggering amyloidogenesis as well as amplification of its detrimental effects in vivo.

Principles of rational design of thermally targeted liposomes for local drug delivery.

Drug release from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes occurs close to the main transition temperature Tm=41°C. The exact release temperature can be adjusted by additional lipids, which shift Tm. A major issue is drug leakage at 37°C. We here describe a novel approach with improved drug retention yet rapid release. To obtain spherical, smooth liposomes we included: i) 2mol% cholesterol, to soften bilayers (Lemmich et al 1997), ii) lipids, which due to their spontaneous curvature stabilize the negative and positive curvatures of the inner and outer leaflets of unilamellar liposomes. In addition to differential scanning calorimetry (DSC) and fluorescence spectroscopy, the lipid mixtures were analyzed by a Langmuir balance for their elastic properties and lipid packing, aiming at high elasticity modulus CS(-1). Maxima in CS(-1) coincided with minima in the free energy of lateral mixing. These liposomes have reduced drug leakage, yet retain rapid release. FROM THE CLINICAL EDITOR: This paper reports the development of optimized DPPC liposomes for drug delivery, with reduced drug leakage but maintained rapid release.

Protein-oxidized phospholipid interactions in cellular signaling for cell death: from biophysics to clinical correlations.

Oxidative stress is associated with several major ailments. However, it is only recently that the developments in our molecular level understanding of the consequences of oxidative stress in modifying the chemical structures of biomolecules, lipids in particular, are beginning to open new emerging insights into the significance of oxidative stress in providing mechanistic insights into the etiologies of these diseases. In this brief review we will first discuss the role of lipid oxidation in controlling the membrane binding of cytochrome c, a key protein in the control of apoptosis. We then present an overview of the impact of oxidized phospholipids on the biophysical properties of lipid bilayers and continue to discuss, how these altered properties can account for the observed enhancement of formation of intermediate state oligomers by cytotoxic amyloid forming peptides associated with pathological conditions as well as host defense peptides of innate immunity. In the third part, we will discuss how the targeting of oxidized phospholipids by i) pathology associated peptides and ii) host defense peptides can readily explain the observed clinical correlations associating Alzheimer's and Parkinson's diseases with increased risk for type 2 diabetes and age-related macular degeneration, and the apparent protective effect of Alzheimer's and Parkinson's diseases from some cancers, as well as the inverse, apparent protection by cancer from Alzheimer's and Parkinson's diseases. This article is part of a Special Issue entitled: Oxidized phospholipids-Their properties and interactions with proteins.

Class specific peptide inhibitors for secretory phospholipases A2.

Phospholipases A2 (PLA2) catalyze the hydrolytic cleavage of free fatty acids from the sn-2 OH-moiety of glycerophospholipids. These enzymes have a number of functions, from digestion to signaling and toxicity of several venoms. They have also been implicated in inflammation and are connected to diverse diseases, such as cancer, ischemia, atherosclerosis, and schizophrenia. Accordingly, there is a keen interest to develop selective inhibitors for therapeutic use. We recently proposed a novel mechanism for the control of PLA2 activity with highly active protofibrils of PLA2 existing transiently before conversion to inactive amyloid fibrils [19]. In keeping with the above mechanism several algorithms identified (85)KMYFNLI(91) and (17)AALSYGFYG(25) in bee venom (bv) and human lacrimal fluid (Lf) PLA2, respectively, as a regions potentially forming amyloid type aggregates. Interestingly, in keeping with the proposed role of these sequences in the control of the activity of these enzymes, preincubation of 2nM bvPLA2 with (85)KMYFNLI(91) caused complete inhibition of PLA2 activity while the scrambled control peptide YNFLIMK had no effect. Approximately 36% attenuation of the hydrolytic activity of LfPLA2 present in human lacrimal fluid was observed in the presence of 80nM (17)AALSYGFYG(25).

Oxidized phosphatidylcholines promote phase separation of cholesterol-sphingomyelin domains.

Lipid lateral segregation in the plasma membrane is believed to play an important role in cell physiology. Sphingomyelin (SM) and cholesterol (Chol)-enriched microdomains have been proposed as liquid-ordered phase platforms that serve to localize signaling complexes and modulate the intrinsic activities of the associated proteins. We modeled plasma membrane domain organization using Langmuir monolayers of ternary POPC/SM/Chol as well as DMPC/SM/Chol mixtures, which exhibit a surface-pressure-dependent miscibility transition of the coexisting liquid-ordered and -disordered phases. Using Brewster angle microscopy and Langmuir monolayer compression isotherms, we show that the presence of an oxidatively modified phosphatidylcholine, 1-palmitoyl-2-azelaoyl-sn-glydecero-3-phosphocholine, efficiently opposes the miscibility transition and stabilizes micron-sized domain separation at lipid lateral packing densities corresponding to the equilibrium lateral pressure of ∼32 mN/m that is suggested to prevail in bilayer membranes. This effect is ascribed to augmented hydrophobic mismatch induced by the oxidatively truncated phosphatidylcholine. To our knowledge, our results represent the first quantitative estimate of the relevant level of phospholipid oxidation that can potentially induce changes in cell membrane organization and its associated functions.

Activation of phospholipase A2 by Hsp70 in vitro.

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer ß-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg(2+).

Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes.

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.

1-Palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, an oxidized phospholipid, accelerates Finnish type familial gelsolin amyloidosis in vitro.

Finnish type familial amyloidosis (FAF) is a neurodegenerative disease, which involves the deposition of D187N or -Y mutant gelsolin fragments as amyloid in various tissues, accompanied by dermatologic, neurologic, and ophthalmologic disorders. Like the other amyloid diseases, FAF is associated with oxidative stress. The latter results in an extensive chemical modification of biomolecules, such as the formation of a myriad of phospholipids with oxidatively modified acyl chains containing various functional groups. Here we demonstrate that 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde moiety at the end of its truncated sn-2 acyl chain, accelerates amyloidogenesis of FtG(179-194) (i.e., the core amyloidogenic segment of residues 179-194 of FAF gelsolin) as revealed by thioflavin T (ThT) fluorescence and electron microscopy. These techniques and Trp fluorescence show that the accelerated conversion of FtG(179-194) into amyloid fibrils consists of distinct consecutive phases. PoxnoPC at a close to critical micelle concentration (~22.5 µM) causes a maximal increase in ThT fluorescence and the K(app) for fibril formation. The rates of fibril elongation and nucleation were proportional to PoxnoPC concentration, while the rates of nucleation were different below and above the critical micelle concentration. Our data also suggest an initial rapid formation of a 1:1 complex by PoxnoPC and FtG(179-194). The latter could involve a transient Schiff base and reside at the membrane hydrocarbon-water interface in the proximity of the phosphocholine headgroup. Subsequently, these profibrils insert into a more hydrophobic milieu and undergo a slow structural transition and assemble into amyloid fibers. Different phases can be expected when proteins aggregate on the phospholipid membrane surfaces, underlying the importance of a detailed kinetic analysis to fully understand the effects of oxidized phospholipids on amyloidogenesis. This study represents the first comprehensive analysis of the kinetics and mechanisms of amyloid formation in the presence of an oxidized phospholipid.

Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine in vitro.

Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90 degrees light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 microM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.

Visualization of intracellular trafficking of Math1 protein in different cell types with a newly-constructed nonviral gene delivery plasmid.

BACKGROUND: In recent years, Math1 gene therapy was indicated to be the future therapy for deafness in combination with other growth factors. However, Math1 delivery using adenovirus-mediated gene delivery or electroporation was impractical. The contribution of Math1 in the combined procedure was not clearly elucidated using the existing plasmids. Nonviral gene delivery vectors are expected to be extremely safe and convenient. The present study aimed to construct the pCDNA6.2/C-EmGFP-Math1 plasmid and evaluate its transfection efficiency and intracellular trafficking of Math1 protein corresponding to transcription regulation function. METHODS: After constructing the pCDNA6.2/C-EmGFP-Math1 expression plasmid, the plasmid was transfected into different cell lines and primary cochlear cells using Lipofectamine 2000. Transfection efficiencies of the plasmid were evaluated. Transfection efficiencies using liposome nanoparticles containing Math1 plasmid were also assessed. Intracellular trafficking of Math1 was monitored using confocal microscopy. RESULTS: Different cell types can be transfected with high transfection efficiencies by the pcDNA6.2/C-EmGFP-Math1 plasmid using Lipofectamine 2000. Liposome nanoparticles containing the Math1 plasmid expressed the gene with variable efficiencies, depending on the particle size, surface charge and PEGylation status. Unique intracellular trafficking of Math1 was demonstrated in different cell types. CONCLUSIONS: The newly-constructed plasmid pcDNA6.2/C-EmGFP-Math1 was suitable for nonviral gene delivery of Math1. Unique intracellular trafficking of Math1 with dynamics from the cytoplasm to the nucleus was demonstrated. The modification of mesenchymal stem cells by Math1 gene delivery and by brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor treatments can potentially be applied to cell replacement for the treatment of cochlear spiral ganglion cell loss in deafness.

Making unilamellar liposomes using focused ultrasound.

Several techniques are available for making large unilamellar vesicles (LUV) with an average diameter of approximately 100 nm, widely employed as model biomembranes as well as vehicles for drug delivery. Here we describe the use of adaptive focused ultrasound (AFU) for the preparation of LUV from multilamellar vesicles (MLV) and studied the effects of ultrasound intensity and number of cycles per burst (CPB) on the average size of vesicles produced. CPB determines the duration of the intermittent pressure wavetrains emitted by the transducer, and the corresponding relaxation periods. Preliminary experiments indicated that CPB controls the size of vesicles assembling after the disruption of MLV by ultrasound and optimum values for obtaining LUV could be iterated. The sizes and lamellarity of LUV were assessed by dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence quenching. AFU provides a simple and easy to use approach for making liposomes with several advantages: it is minimally invasive and involves no loss of material. Precisely controlled wavelengths are employed with a significant reduction in the presence of hot spots, which could destroy some biological materials of interest.

Cytochrome c-lipid interactions: new insights from resonance energy transfer.

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥ 40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.