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BBA - Biomembranes (v.1711, #1)
Biophysical characterization of anionic lipoplexes by Siddhesh D. Patil; David G. Rhodes; Diane J. Burgess (pp. 1-11).
Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca2+ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca2+–DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca2+-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca2+ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca2+ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.
Keywords: Anionic lipoplex; Transfection; DNA delivery; Anionic liposome; DNA conformation
Molecular associations and surface-active properties of short- and long- N-acyl chain ceramides by Jesús Sot; Félix M. Goñi; Alicia Alonso (pp. 12-19).
The behaviour of N-hexadecanoylsphingosine (Cer16), N-hexanoylsphingosine (Cer6) and N-acetylsphingosine (Cer2) in aqueous media and in lipid–water systems, monolayers and bilayers has been comparatively examined using Langmuir balance and fluorescence techniques. Cer16 behaves as an insoluble non-swelling amphiphile, not partitioning into the air–water interface, thus not modifying the surface pressure of the aqueous solutions into which it is included. By contrast both Cer6 and Cer2 behave as soluble amphiphiles, up to approx. 100 μM. At low concentrations, they become oriented at the air–water interface, increasing surface pressure in a dose-dependent way up to ca. 5 μM bulk concentration. At higher concentrations, the excess ceramide forms micelles, critical micellar concentrations of both Cer6 and Cer2 being in the 5–6 μM range. When the air–water interface is occupied by a phospholipid, 6Cer2 and Cer6 become inserted in the phospholipid monolayer, causing a further increase in surface pressure. This increase is dose dependent, and reaches a plateau at ca. 2 μM ceramide bulk concentration. Both Cer2 and Cer6 become inserted in phospholipid monolayers with initial surface pressures of up to 43 and 46 mN m−1, respectively, which ensures their capacity to become inserted into cell membranes whose monolayers are estimated to support a surface pressure of about 30 mN m−1. Both Cer2 and Cer6, but not Cer16, had detergent-like properties, such as giving rise to phospholipid–ceramide mixed micelles, when added to phospholipid monolayers or bilayers. The short-chain ceramides form large aggregates and precipitate at concentrations above approx. 100 μM. These results are relevant in cell physiology studies in which short- and long-chain ceramides are sometimes used as equivalent molecules, in spite of their different biophysical behaviour.
Keywords: Abbreviations; ANS; 1-anilinonaphthalene-8-sulfonic acid; Cer2; N; -acetylsphingosine; Cer6; N; -hexanoylsphingosine; Cer16; N; -hexadecanoylsphingosine; cmc; critical micellar concentration; DEPE; dielaidoylphosphatidylethanolamine; DMSO; dimethylsulfoxide; MLV; multilamellar vesicles; NBD-SM; 6-((; N; -(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl)sphingosylphosphocholine; PC; phosphatidylcholine; Rh-PE; N; -(lissamine rhodamine B sulfonyl) phosphatidylethanolamineCeramide; Short-chain ceramide; Amphiphile; Surfactant; Detergent; Lipid monolayer; Surface pressure; Bilayer solubilization; Micelle
Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells by Akira Ikari; Hitoshi Harada; Kuniaki Takagi (pp. 20-24).
We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin–Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [14C]-α-methyl glucopyranoside ([14C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [14C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.
Keywords: Green fluorescent protein; Protein kinase A; SGLT1
Liposomes targeted via two different antibodies: Assay, B-cell binding and cytotoxicity by Kimberley Laginha; Davis Mumbengegwi; Theresa Allen (pp. 25-32).
The selective toxicity of anticancer drugs can be improved with the use of antibody-targeted liposomes. We hypothesize that liposomes targeted via antibodies against two or more receptor populations will increase the apparent receptor density on the target cells, resulting in improved therapeutic affects. A fluorescent assay was developed, using the fluorophores Alexa Fluor® 350 and 532 to label monoclonal antibodies (mAb), and used to quantitate two different mAb populations coupled to the same liposome surface to within ±10% of the values obtained with radiolabeled antibody (125I) tracers. The binding and uptake of targeted liposomes by B lymphoma (Namalwa) cells were examined for either individual populations of αCD19-targeted or αCD20-targeted liposomes, mixed populations (1:1) of αCD19-targeted liposomes plus αCD20-targeted liposomes, and dual-targeted liposomes, i.e., equal amount of both αCD19 and αCD20 on the same liposomes. At similar antibody densities, the binding and uptake of the dual-targeted liposomes were greater than that of either individually targeted liposomes alone, and showed additivity. At the same total lipid and antibody densities, 1:1 mixtures of individually targeted liposomes gave similar results to dual-targeted liposomes. Cytotoxicity was also improved, with DXR-loaded dual-targeted liposomes appearing to have higher cytotoxicity than 1:1 mixtures of individually targeted liposomes.
Keywords: Abbreviations; DXR; doxorubicin; mAb; monoclonal antibody; SL; non-targeted long-circulating (Stealth®) liposomes; SIL; long-circulating antibody-targeted liposomes (Stealth® immunoliposomes); αCD19; anti-CD19 mAb; αCD20; anti-CD20 mAb (Rituxan); HSPC; hydrogenated soy phosphatidylcholine; CHOL; cholesterol; mPEG; 2000; -DSPE; methoxy poly(ethylene glycol) (M; r; 2000) covalently linked to distearoylphosphatidylethanoloamine; Mal-PEG-DSPE; maleimide-derivatized PEG-DSPE; [; 3; H]CHE; Chol-[1,2-; 3; H-(; N; )]hexadecyl ether; MTT; 3-(4,5-dimethyltiazol-2-ly)-2,5-diphenyltetrazolium bromide; FITC; fluorescein isothiocyanate; HBS; HEPES-buffered saline; PL; phospholipid; SIL[αCD19]; anti-CD19-targeted immunoliposomes; SIL[αCD20]; anti-CD20-targeted immunoliposomes; SIL[αCD19]+SIL[αCD20]; 1:1 mixtures of individual immunoliposomes; SIL[αCD19+αCD20]); dual-targeted immunoliposomesAntibody-mediated targeting; Immunoliposome; Anti-CD19; Anti-CD20; B-cell lymphoma; Liposomal doxorubicin
Store-operated calcium entry in differentiated C2C12 skeletal muscle cells by Yolanda Gutierrez-Martin; Francisco Javier Martin-Romero; Fernando Henao (pp. 33-40).
In this paper, we show further evidences for the existence of store-operated calcium entry in differentiated skeletal muscle C2C12 myotubes after Ca2+ depletion in sarcoplasmic reticulum, using thapsigargin, a potent sarcoplasmic reticulum Ca2+-ATPase inhibitor, caffeine as ryanodine receptor activator, and ATP which activates purinergic receptors. The quenching of fura 2 fluorescence emission by Mn2+ also provided evidences for store-operated calcium entry because this quenching was accelerated when sarcoplasmic reticulum was depleted of Ca2+. Ca2+ entry was sensitive to Ni2+, La3+, Gd3+ and 2-aminoethyl diphenyl borate but resistant to nifedipine, thus excluding L-type Ca2+ channels in this type of calcium entry. Our data obtained using ATP for store depletion suggest that the level of Ca2+ in internal stores could play a role in the regulation of store-operated calcium channel activity in this cell type.
Keywords: Abbreviations; 2-APB; 2-aminoethyl diphenyl borate; [Ca; 2+; ]; i; intracellular free calcium concentration; Fura 2-AM; fura 2-acetoxymethylester; IP; 3; inositol-1,4,5-trisphosphate; IP; 3; R; IP; 3; receptor; PMSF; phenylmethylsulfonyl fluoride; RyR; ryanodine receptor; SOCC; store-operated calcium channel; SOCE; store-operated calcium entry; SR; sarcoplasmic reticulum; TG; thapsigarginCapacitative calcium entry; Store-operated calcium entry; Skeletal muscle; C2C12 myotube; Calcium; Fura 2
Electroneutral K+/H+ exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins by Elisabeth Froschauer; Karin Nowikovsky; Rudolf J. Schweyen (pp. 41-48).
YOL027c in yeast and LETM1 in humans encode integral proteins of the inner mitochondrial membrane. They have been implicated in mitochondrial K+ homeostasis and volume control. To further characterize their role, we made use of submitochondrial particles (SMPs) with entrapped K+- and H+-sensitive fluorescent dyes PBFI and BCECF, respectively, to study the kinetics of K+ and H+ transport across the yeast inner mitochondrial membrane. Wild-type SMPs exhibited rapid, reciprocal translocations of K+ and H+ driven by concentration gradients of either of them. K+ and H+ translocations have stoichiometries similar to those mediated by the exogenous K+/H+ exchanger nigericin, and they are shown to be essentially electroneutral and obligatorily coupled. Moreover, [K+] gradients move H+ against its concentration gradient, and vice-versa. These features, as well as the sensitivity of K+ and H+ fluxes to quinine and Mg2+, qualify these activities as K+/H+ exchange reactions. Both activities are abolished when the yeast Yol027p protein is absent ( yol027Δ mutant SMPs), indicating that it has an essential role in this reaction. The replacement of the yeast Yol027p by the human Letm1 protein restores K+/H+ exchange activity confirming functional homology of the yeast and human proteins. Considering their newly identified function, we propose to refer to the yeast YOL027c gene and the human LETM1 gene as yMKH1 and hMKH1, respectively.
Keywords: Abbreviations; DCCD; Dicyclohexylcarbodiimide; SMP; submitochondrial particle; ΔΨ; membrane potentialSubmitochondrial particle; BCECF; PBFI; Cation/proton exchange; Inner mitochondrial membrane; K; +; /H; +; antiport
Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843 by Sathiah Thennarasu; Dong-Kuk Lee; Anmin Tan; U. Prasad Kari; Ayyalusamy Ramamoorthy (pp. 49-58).
Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct–OOLLOOLOOL–NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide.2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers.31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid–peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.
Keywords: Abbreviations; CSA; chemical shift anisotropy; CD; circular dichroism; DiPoPE; 1,2-dipalmitoleoyl-; sn; -glycero-3-phosphotydylethanolamine; DSC; differential scanning calorimetry; H; I; normal hexagonal phase; H; II; inverted hexagonal phase; ITC; isothermal titration calorimetry; L; α; fluid lamellar phase; MIC; minimum inhibitory concentration; MLVs; multilamellar vesicles; NMR; nuclear magnetic resonance; O; ornithine; Oct; octanoyl; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPC-d; 31; 1-d; 31; -palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylglycerol; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylethanolamine; SUVs; small unilamellar vesiclesLipopeptide; Antimicrobial activity; Membrane permeabilization; Solid-state NMR; Oriented bilayers; Curvature strain
Raman spectra of planar supported lipid bilayers by Chongsoo Lee; Colin D. Bain (pp. 59-71).
Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C–H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14–41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (Lα–Lβ') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.
Keywords: Total internal reflection; Raman spectroscopy; Phase transition; Planar supported lipid bilayer; Dimyristoyl phosphatidylcholine; Dipalmitoyl phosphatidylcholine
Electrostatic basis of valence selectivity in cationic channels by Ben Corry; Taira Vora; Shin-Ho Chung (pp. 72-86).
We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.
Keywords: Ion channel; Selectivity; Sodium channel; Calcium channel; Potassium channel; Brownian dynamic; Block; Electrostatic
Differential effect of phosphatidylethanolamine depletion on raft proteins by Miroslava Opekarová; KateÅ™ina MalÃnská; Linda Nováková; Widmar Tanner (pp. 87-95).
A considerable amount of evidence supports the idea that lipid rafts are involved in many cellular processes, including protein sorting and trafficking. We show that, in this process, also a non-raft lipid, phosphatidylethanolamine (PE), has an indispensable function. The depletion of this phospholipid results in an accumulation of a typical raft-resident, the arginine transporter Can1p, in the membranes of Golgi, while the trafficking of another plasma membrane transporter, Pma1p, is interrupted at the level of the ER. Both these transporters associate with a Triton (TX-100) resistant membrane fraction before their intracellular transport is arrested in the respective organelles. The Can1p undelivered to the plasma membrane is fully active when reconstituted to a PE-containing vesicle system in vitro. We further demonstrate that, in addition to the TX-100 resistance at 4 °C, Can1p and Pma1pa exhibit different accessibility to nonyl glucoside (NG), which points to distinct intimate lipid surroundings of these two proteins. Also, at 20 °C, these two proteins are extracted by TX-100 differentially. The features above suggest that Pma1p and Can1p are associated with different compartments. This is independently supported by the observations made by confocal microscopy. In addition we show that PE is involved in the stability of Can1p–raft association.
Keywords: Lipid raft; Trafficking; Saccharomyces cerevisiae; Can1p; Pma1p