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BBA - Biomembranes (v.1768, #5)

Editorial Board (pp. ii).

How much solute is needed to inhibit the fluid to gel membrane phase transition at low hydration? by Thomas Lenné; Gary Bryant; Roland Holcomb; Karen L. Koster (pp. 1019-1022).

We present a quantitative study of the effect of sugars on the membrane gel–fluid phase transition as a function of sugar:lipid ratio. We show that the maximum effect occurs at around 1.5 sugar rings per molecule for both mono- and di-saccharides. We present a theoretical model to try to explain these results, and discuss the assumptions inherent in the model.

Keywords: Phospholipid; Dehydration; Sugar; Desiccation; Freezing; Lipid phase transition

Different ionic conditions prompt NHE2 and NHE3 translocation to the plasma membrane by J. Scott Gens; Hongwei Du; Lixuan Tackett; Shen-Shen Kong; Shaoyou Chu; Marshall H. Montrose (pp. 1023-1035).

We tested whether NHE3 and NHE2 Na⁺/H⁺ exchanger isoforms were recruited to the plasma membrane (PM) in response to changes in ion homeostasis. NHE2-CFP or NHE3-CFP fusion proteins were functional Na⁺/H⁺ exchangers when transiently expressed in NHE-deficient PS120 fibroblasts. Confocal morphometry of cells whose PM was labeled with FM4-64 measured the fractional amount of fusion protein at the cell surface. In resting cells, 10–20% of CFP fluorescence was at PM and stable over time. A protocol commonly used to activate the Na⁺/H⁺ exchange function (NH₄-prepulse acid load sustained in Na⁺-free medium), increased PM percentages of PM NHE3-CFP and NHE2-CFP. Separation of cellular acidification from Na⁺ removal revealed that only NHE3-CFP translocated when medium Na⁺ was removed, and only NHE2-CFP translocated when the cell was acidified. NHE2/NHE3 chimeric proteins demonstrate that the Na⁺-removal response element resides predominantly in the NHE3 cytoplasmic tail and is distinct from the acidification response sequence of NHE2.

Keywords: Abbreviations; Na; _o; ⁺; external sodium concentration; pH; _i; intracellular pH; PM; plasma membrane; TMA; tetramethylammonium; V; _max; maximum velocity of transportCell membrane; Hydrogen-ion concentration; Sodium; Exocytosis; Endocytosis

Distribution of reaction products in phospholipase A₂ hydrolysis by Hanna P. Wacklin; Fredrik Tiberg; Giovanna Fragneto; Robert K. Thomas (pp. 1036-1049).

We have monitored the composition of supported phospholipid bilayers during phospholipase A₂ hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA₂ shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5±3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA₂, which resides in a 21±1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d₃₁-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA₂ molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA₂.

Keywords: Abbreviations; d; ₃₁; -POPC; l; -α-1-; O; -d; ₃₁; -palmitoyl,2-; O; -oleyl,3-; O; -; sn; -glycerophosphocholine; PLA; ₂; phospholipase A; ₂; DOPC; l; -α-1,2-; O; -dioleyl-3-; O; -; sn; -glycerophosphocholine; DPPC; l; -α-1,2-; O; - palmitoyl-3-; O; -; sn; -glycerophosphocholine; DDM; n-β-; d; -docecyl maltosidePhospholipase A; ₂; Lag phase; Lyso-lipid; Fatty acid; Supported bilayer; Neutron reflection

The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes by K. Rieber; J. Sýkora; A. Olżyńska; R. Jelinek; G. Cevc; M. Hof (pp. 1050-1058).

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Δν, and the time course of the correlation function, C( t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.

Keywords: Time-resolved fluorescence; Mixed lipid/surfactant vesicles; Tween 80; Brij 98; Interferon alfa-2b; Melittin

Identification of a segment in the precursor of pulmonary surfactant protein SP-B, potentially involved in pH-dependent membrane assembly of the protein by Alicia G. Serrano; Elisa J. Cabré; Jesús Pérez-Gil (pp. 1059-1069).

In the present work, the hydrophobic properties of proSP-B, the precursor of pulmonary surfactant protein SP-B, have been analyzed under different pH conditions, and the sequence segment at position 111–135 of the N-terminal domain of the precursor has been detected as potentially possessing pH-dependent hydrophobic properties. We have studied the structure and lipid–protein interactions of the synthetic peptides BpH, with sequence corresponding to the segment 111–135 of proSP-B, and BpH-W, bearing the conservative substitution F127W to use the tryptophan as an intrinsic fluorescent probe. Peptide BpH-W interacts with both zwitterionic and anionic phospholipid vesicles at neutral pH, as monitored by the blue-shifted maximum emission of its tryptophan reporter. Insertion of tryptophan into the membranes is further improved at pH 5.0, especially in negatively-charged membranes. Peptides BpH and BpH-W also showed pH-dependent properties to insert into phospholipid monolayers. We have also found that the single sequence variation F120K decreases substantially the interaction of this segment with phospholipid surfaces as well as its pH-dependent insertion into deeper regions of the membranes. We hypothesize that this region could be involved in pH-triggered conformational changes occurring in proSP-B along the exocytic pathway of surfactant in type II cells, leading to the exposure of the appropriate segments for processing and assembly of SP-B within surfactant lipids.

Keywords: Lung surfactant; SP-B; Lipid–protein interactions; Synthetic peptides; Interfacial hydrophobicity; Secretory pathway

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B_1–25 and SP-B_8–25 by Nilanjana Biswas; Alan J. Waring; Frans J. Walther; Richard A. Dluhy (pp. 1070-1082).

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1–25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1–25 was compared with that of a truncated homodimer (dSP-B_8–25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S–S stretching region centered at ∼500 cm−¹ indicated a stable, although highly strained disulfide conformation with a χ(CS–SC) dihedral angle of ±10° for the dSP-B_1–25 dimer. In contrast, the truncated dimer dSP-B_8–25 exhibited a series of disulfide conformations with the χ(CS–SC) dihedral angle taking on values of either ±30° or 85±20°. For conformations with χ(CS–SC) close to the ±90° value, the Raman spectra of the 8–25 truncated dimers exhibited χ(SS–CC) dihedral angles of 90/180° and 20–30°. In the presence of a lipid mixture, both constructs showed a ν(S–S) band at ∼488 cm−¹, corresponding to a χ(CS–SC) dihedral angle of ±10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼60–70° to the surface normal, while the β structure had ∼40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.

Keywords: Abbreviations; ARDS; acute respiratory distress syndrome; ATR; attenuated total reflectance; A/W; air–water; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DOPG; 1,2-dioleoyl-; sn; -glycero-3-[phosphor-; rac; -(1-glycerol)](sodium salt); HMP; 4-hydroxymethylphenoxyacetyl-4′-methylbenzyhydrylamine resin; RDS; respiratory distress syndrome; SP-B; pulmonary surfactant protein B; SP-B; _1–25; synthetic peptide containing the first 25 amino acids of the N-terminal sequence of SP-B; SP-B; _8–25; synthetic peptide containing amino acids 8–25 of the N-terminal sequence of SP-B; TFA; trifluoroacetic acid; TFE; trifluoroethanoldsp-b; _1–25; dsp-b; _8–25; Surfactant protein (sp)-b; Attenuated total reflectance IR spectroscopy; Raman spectroscopy; Lung surfactant; Synthetic peptide

Cellular and molecular effects of unoprostone as a BK channel activator by John Cuppoletti; Danuta H. Malinowska; Kirti P. Tewari; Jayati Chakrabarti; Ryuji Ueno (pp. 1083-1092).

Effects of unoprostone isopropyl (unoprostone), a prostaglandin metabolite analog; latanoprost, a PGF_2α analog; and PGF_2α were examined in HCN-1A cells, a model system for studies of large conductance Ca²⁺ activated K⁺(BK) channel activator-based neuroprotective agents. Unoprostone and latanoprost, both used as anti-glaucoma agents, have been suggested to act through FP receptors and have neuroprotective effects. Ion channel activation, plasma membrane polarization, [Ca²⁺]_i changes and protection against long-term irreversible glutamate-induced [Ca²⁺]_i increases were studied. Unoprostone activated iberiotoxin (IbTX)-sensitive BK channels in HCN-1A cells with an EC₅₀ of 0.6±0.2 nM and had no effect on Cl⁻ currents. Unoprostone caused IbTX-sensitive plasma membrane hyperpolarization that was insensitive to AL8810, an FP receptor antagonist. In contrast, latanoprost and PGF_2α activated a Cl⁻ current sensitive to [Ca²⁺]_i chelation, tamoxifen and AL8810, and caused IbTX-insensitive, AL8810-sensitive membrane depolarization consistent with FP receptor-mediated Ca²⁺ signaling Cl⁻ current activation. Latanoprost and PGF_2α, but not unoprostone, increased [Ca²⁺]_i. Unoprostone, PGF_2α only partially, but not latanoprost protected HCN-1A cells against glutamate-induced Ca²⁺ deregulation. These findings show that unoprostone has a distinctly different mechanism of action from latanoprost and PGF_2α. Whether unoprostone affects the BK channel directly or an unidentified signaling mechanism has not been determined.

Keywords: HCN-1A; Latanoprost; PGF; _2α; IbTX; Neuroprotection; Glaucoma

Evaluation of the antitumoral effect mediated by IL-12 and HSV-tk genes when delivered by a novel lipid-based system by H. Faneca; A.S. Cabrita; S. Simões; M.C. Pedroso de Lima (pp. 1093-1102).

In the present work, we used a novel albumin-associated lipoplex formulation, containing the cationic lipid 1-palmitoyl-2-oleoyl- sn-glycero-3-ethylphosphocholine (EPOPC) and cholesterol (Chol), to evaluate the antitumoral efficacy of two gene therapy strategies: immuno-gene therapy, mediated by IL-12 gene expression, and “suicide” gene therapy, mediated by HSV-tk gene expression followed by ganciclovir (GCV) treatment. Our data show that, in an animal model bearing a subcutaneous TSA (mouse mammary adenocarcinoma) tumor, intratumoral administration of the albumin-associated complexes containing the plasmid encoding IL-12 results in a strong antitumoral effect, as demonstrated by the smaller tumor size, the higher T-lymphocyte tumor infiltration and the more extensive tumor necrotic and hemorrhagic areas, as compared to that observed in animals treated with control complexes. On the other hand, the application of the “suicide” gene therapy strategy results in a significant antitumoral activity, which is similar to that achieved with the immuno-gene therapy strategy, although involving different antineoplastic mechanisms. For the tested model, albumin-associated complexes were shown to efficiently mediate intratumoral delivery of therapeutic genes, thus leading to a significant antitumoral effect. This finding is particularly relevant since TSA tumors are characterized for being poorly immunogenic, aggressive and exhibiting high proliferation capacity.

Keywords: Cancer gene therapy; “Suicide” gene therapy; Immunotherapy; Gene delivery; Cationic liposomes; Transfection

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers by Devaki A. Kelkar; Amitabha Chattopadhyay (pp. 1103-1113).

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831–843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.

Keywords: Abbreviations; DAPC; 1,2-diarachidoyl-; sn; -glycero-3-phosphocholine [diC20:0 PC]; DCPC; 1,2-Dicapryl-; sn; -glycero-3-phosphocholine [diC10:0 PC]; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphocholine [diC12:0 PC]; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine [diC14:0 PC]; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine [diC16:0 PC]; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine [diC18:0 PC]; MLV; multilamellar vesicle; REES; red edge excitation shift; SUV; small unilamellar vesicleGramicidin; Hydrophobic mismatch; REES; Acrylamide quenching

Functional incorporation of the pore forming segment of AChR M2 into tethered bilayer lipid membranes by Inga K. Vockenroth; Petia P. Atanasova; Joanna R. Long; A. Toby A. Jenkins; Wolfgang Knoll; Ingo Köper (pp. 1114-1120).

Tethered bilayer lipid membranes (tBLMs) are robust and flexible model platforms for the investigation of various membrane related processes. They are especially suited to study the incorporation and function of ion channel proteins, where a high background resistance of the membrane is essential. Synthetic M2 peptides, analogues of the transmembrane fragment of the acetylcholine receptor, could be incorporated into two different membrane architectures. The functional reconstitution and the formation of a conducting pore are shown by electrochemical impedance spectroscopy (EIS). The pore is selective for small monovalent cations, while bulky ions cannot pass. This is a significant step towards a novel biosensing approach. We envision a device, where a stable and insulating membrane would be attached to an electronic read-out unit and embedded proteins would serve as actual sensing units.

Characterization of the drug retention and pharmacokinetic properties of liposomal nanoparticles containing dihydrosphingomyelin by Michael J.W. Johnston; Sean C. Semple; Sandy K. Klimuk; Steve Ansell; Norbert Maurer; Pieter R. Cullis (pp. 1121-1127).

The drug retention and circulation lifetime properties of liposomal nanoparticles (LN) containing dihydrosphingomyelin (DHSM) have been investigated. It is shown that replacement of egg sphingomyelin (ESM) by DHSM in sphingomyelin/cholesterol (Chol) (55/45; mol/mol) LN results in substantially improved drug retention properties both in vitro and in vivo. In the case of liposomal formulations of vincristine, for example, the half-times for drug release ( T_1/2) were approximately 3-fold longer for DHSM/Chol LN as compared to ESM/Chol LN, both in vitro and in vivo. Further increases in T_1/2 could be achieved by increasing the drug-to-lipid ratio of the liposomal vincristine formulations. In addition, DHSM/Chol LN also exhibit improved circulation lifetimes in vivo as compared to ESM/Chol LN. For example, the half-time for LN clearance (Tc_1/2) at a low lipid dose (15 μmol lipid/kg, corresponding to 8 mg lipid/kg body weight) in mice was 3.8 h for ESM/Chol LN compared to 6 h for DHSM/Chol LN. In addition, it is also shown that DHSM/Chol LN exhibit much longer half-times for vincristine release as compared to LN with the “Stealth” lipid composition. It is anticipated that DHSM/Chol LN will prove useful as drug delivery vehicles due to their excellent drug retention and circulation lifetime properties.

Keywords: Abbreviations; SM; sphingomyelin; ESM; Egg sphingomyelin; DHSM; dihydrosphingomyelin; BSM; bovine brain sphingomyelin; MSM; bovine milk sphingomyeline; HSPC; Hydrogenated Soybean phosphatidlycholine; DSPE-PEG; ₂₀₀₀; Distearoyl-; sn; -Glycero-3-Phosphoethanolamine-N-[Poly(ethyleneglycol)2000]; αTC; α-tocopherol; [; ³; H]-CHE; [; ³; H]-cholesterylhexadecyl ether; MLV; multilamellar vesicles; LN; liposomal nanoparticleLiposomes; Vincristine; Regulated drug release; Dihydrosphingomyelin; Liposomal pharmacokinetics

GM1-ganglioside-induced Aβ assembly on synaptic membranes of cultured neurons by Naoki Yamamoto; Yuko Fukata; Masaki Fukata; Katsuhiko Yanagisawa (pp. 1128-1137).

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid β-protein (Aβ). A marked Aβ assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Aβ assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Aβ assembly in the culture was completely suppressed by the coincubation of Aβ with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Aβ (GAβ). In primary neuronal cultures, Aβ assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Aβ assembly through the generation of GAβ, which is an endogenous seed for Aβ assembly in Alzheimer brain.

Keywords: Alzheimer's disease; Amyloid; Amyloid β-protein; Seed; Ganglioside; Synapse; PC12 cell; Primary neuron

Interaction of piroxicam with mitochondrial membrane and cytochrome c by Hirak Chakraborty; Prabir K. Chakraborty; Sanghamitra Raha; Parikshit C. Mandal; Munna Sarkar (pp. 1138-1146).

Modulation of surface properties of biomembranes by any ligand leading to permeabilization, fusion, rupture, etc. is a fundamental requirement for many biological processes. In this work, we present the interaction of piroxicam, a long acting Non-Steroidal Anti-Inflammatory Drug (NSAID) with isolated mitochondria, membrane mimetic systems, intact cells and a mitochondrial protein cytochrome c. Dye permeabilization study on isolated mitochondria indicates that piroxicam can permeabilize mitochondrial membrane. Direct imaging by Scanning Electron Microscope (SEM) shows that piroxicam induces changes in mitochondrial membrane morphology leading to fusion and rupture. Transmission Electron Microscope (TEM) imaging of piroxicam treated DMPC vesicles and mixed micelles formed from CTAB and SDS show that causing membrane fusion is a general property of piroxicam at physiological pH. In intact cells viz., V79 Chinese Hamster lung fibroblast, piroxicam is capable of releasing cytochrome c from mitochondria into the cytosol in a dose dependent manner along with the enhancement of downstream proapoptotic event viz., increase in caspase-3 activity. We have also shown that piroxicam can reduce cytochrome c within a time frame relevant to its lifetime in blood plasma. UV-visible spectroscopy has been used to study the reaction mechanism and kinetics in detail, allowing us to propose and validate a Michaelis–Menten like reaction scheme. CD spectroscopy shows that small but significant changes occur in the structure of cytochrome c when reduced by piroxicam.

Keywords: Abbreviations; NSAID; Nonsteroidal anti-inflammatory drug; SEM; Scanning electron microscope; TEM; Transmission electron microscope; DMPC; Dimyristoyl phosphatidylcoline; COX; Cyclooxygenase; MOPS; 3-Morpholinopropane sulphonic acid; DMSO; Dimethyl sulfoxide; PBS; Phosphate buffer saline; EGTA; Ethylene glycol-bis(β-aminoethyl ether)-; N,N,N′,N′; -tetraacetic acid; EDTA; Ethylenediaminetetraaceticacid; PTA; Phosphotungstic acid; CD; Circular dichroism; PMSF; Phenylmethylsulfonyl fluoride; PVDF; Polyvinylidene difluorideNSAIDs; Mitochondria; Membrane fusion; Cytochrome; c; Kinetics; Optical spectroscopy

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species? by V. Battaglia; C.A. Rossi; S. Colombatto; M.A. Grillo; A. Toninello (pp. 1147-1153).

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H₂O₂ generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H₂O₂ and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron–sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca²⁺. Instead, 1 mM agmatine generates larger amounts of H₂O₂ than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca²⁺-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.

Keywords: Abbreviations; ADC; arginine decarboxylase; AGM; agmatine; BHT; butylhydroxytoluene; CsA; cyclosporin A; DMO; 5,5′-dimethyl-oxazolidine-2,4-dione; MAO; monoamine oxidase; MPT; mitochondrial permeability transition; NEM; N-ethylmaleimide; NOS; nitric oxide synthase; RLM; rat liver mitochondria; ROS; reactive oxygen species; TPP; ⁺; tetraphenylphosphonium; Δ; Ψ; membrane potentialAgmatine; Mitochondria; Reactive oxygen species; Permeability transition

Expression and functionality of the Na⁺/ myo-inositol cotransporter SMIT2 in rabbit kidney by Karim Lahjouji; Rym Aouameur; Pierre Bissonnette; Michael J. Coady; Daniel G. Bichet; Jean-Yves Lapointe (pp. 1154-1159).

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a “compatible osmolyte”. Currently, three MI cotransporters have been identified: two are Na⁺-dependent (SMIT1 and SMIT2) and one is H⁺-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence ofd- chiro-inositol andl-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.

Keywords: Myo-inositol transport; Membrane transport; SMIT2; Rabbit renal BBMV

Action mechanism of tachyplesin I and effects of PEGylation by Yuichi Imura; Minoru Nishida; Yoshiyuki Ogawa; Yoshinobu Takakura; Katsumi Matsuzaki (pp. 1160-1169).

PEGylation of protein and peptide drugs is frequently used to improve in vivo efficacy. We investigated the action mechanism of tachyplesin I, a membrane-acting cyclic antimicrobial peptide from Tachypleus tridentatus and the effects of PEGylation on the mechanism. The PEGylated peptide induced the leakage of calcein from egg yolkl-α-phosphatidylglycerol/egg yolkl-α-phosphatidylcholine large unilamellar vesicles similarly to the parent peptide. Both peptides induced lipid flip-flop coupled to leakage and was translocated into the inner leaflet of the bilayer, indicating that tachyplesin I forms a toroidal pore and that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. Despite their similar activities against model membranes, the peptides showed very different biological activities. The cytotoxicity of tachyplesin I was greatly reduced by PEGylation, although the antimicrobial activity was significantly weakened. We investigated the enhancement of the permeability of inner membranes induced by the peptides. Our results suggested that outer membranes and peptidoglycan layers play an inhibitory role in the permeation of the PEG moiety. Furthermore, a reduction in DNA binding by PEGylation may also contribute to the weak activity of the PEGylated peptide.

Keywords: Abbreviations; AMEM; Alpha modification of Eagle's medium; C; ₆; -NBD-PC; 1-palmitoyl-2-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]caproyl]-; l; -α-phosphatidylcholine; CD; circular dichroism; CFU; colony forming unit; CHO; Chinese Hamster Ovary; DNS-PE; N; -(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine, triethylammonium salt; FBS; fetal bovine serum; Fmoc; 9-fluorenylmethoxycarbonyl; HPLC; high-performance liquid chromatography; L; /; P; lipid-to-peptide molar ratio; LUVs; large unilamellar vesicles; MIC; minimal inhibitory concentration; MLVs; multilamellar vesicles; NAPB; 10 mM sodium phosphate/100 mM NaCl, pH 7.4 buffer; ONPG; o; -nitrophenyl-β-; d; -galactoside; PC; egg yolk; l; -α-phosphatidylcholine; PEG; polyethyleneglycol; PG; l; -α-phosphatidyl-; dl; -glycerol enzymatically converted from PC; Pyrene-PC; 1-hexadecanoyl-2-(1-pyrenehexanoyl)-; sn; -glycero-3-phosphocholine; SUVs; small unilamellar vesicles; TSB; tryptic soy broth; WST-1; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-; 2H; -tetrazolium, monosodium saltAntimicrobial peptide; Tachyplesin I; PEGylation; Lipid flip-flop; Peptide–lipid interaction; Membrane permeabilization

Association of putative ammonium exporters Ato with detergent-resistant compartments of plasma membrane during yeast colony development: pH affects Ato1p localisation in patches by Markéta Řičicová; Helena Kučerová; Libuše Váchová; Zdena Palková (pp. 1170-1178).

It was proposed that Ato1p, Ato2p and Ato3p have a role in ammonia production by Saccharomyces cerevisiae colonies (Palkova et al., Mol Biol Cell 13: 3901–3914, 2002). In this study, we show that all three Ato proteins localise to the plasma membrane and their appearance correlates with the beginning of ammonia release. The expression of ATO genes is controlled by ammonia. All three Ato–GFP proteins associate with detergent-resistant membranes; two of them, Ato1p–GFP and Ato3p–GFP, localise to patches visible under the fluorescence microscope. In contrast with Ato3p–GFP which forms stable patches, the formation of those of Ato1p–GFP is pH dependent. Ato1p–GFP patches form at pH above 6 and they disappear at pH 5 or lower. Both changes, Ato1p–GFP clustering and patches spreading are reversible. The Ato1p–GFP spreading at low pH is independent on endocytosis. These data suggest that besides the ammonia induction of Ato protein synthesis, pH may rapidly regulate Ato1p function.

Keywords: Abbreviations; BKP; bromcresol purple; DRM; detergent-resistant membrane; TMD; transmembrane domain Saccharomyces cerevisiae; colony; Ammonia signalling; Ato protein production; Detergent resistant membrane; Endocytosis

The response of giant phospholipid vesicles to pore-forming peptide melittin by Mojca Mally; Janja Majhenc; Saša Svetina; Boštjan Žekš (pp. 1179-1189).

The interaction between the pore-forming peptide melittin (MLT) and giant phospholipid vesicles was explored experimentally. Micromanipulation and direct optical observation of a vesicle (loaded with sucrose solution and suspended in isomolar glucose solution) enabled the monitoring of a single vesicle response to MLT. Time dependences of the vesicle size, shape and the composition of the inner solution were examined at each applied concentration of MLT (in the range from 1 to 60 μg/ml). The response varied with MLT concentration from slight perturbation of the membrane to disintegration of the vesicle. A model for MLT–vesicle interaction is proposed that explains the observed phenomena in the entire span of MLT concentrations and is consistent with deduced underlying mechanisms of MLT action: trans-membrane positioning and dimerization of MLT, the lipid flow from the outer to the inner membrane leaflet induced by MLT translocation, formation of pores and the consequent transport of small molecules through the membrane. The results of the theoretical analysis stress the role of dimers in the MLT–membrane interaction and demonstrate that the MLT-induced membrane permeability for sugar molecules in this experimental set-up depends on both MLT concentration and time.

Keywords: Membrane permeability; Tension pores; Directed lipid flow; Lysis; Dimers

Atomic force microscopy and force spectroscopy study of Langmuir–Blodgett films formed by heteroacid phospholipids of biological interest by Sergi Garcia-Manyes; Òscar Domènech; Fausto Sanz; M.Teresa Montero; Jordi Hernandez-Borrell (pp. 1190-1198).

Langmuir–Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with χ_POPC=0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m−¹ revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force–extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with χ_POPC=0.4 the jump occurs at ∼800 pN. Widths of ∼2 nm could be established for POPC and χ_POPC=0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC–LC′) occurring at pressures >36.5 mN m−¹. This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force (∼1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.

Keywords: Atomic force Microscopy (AFM); Force spectroscopy (FS); Langmuir–Blodgett film (LBs)

Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: Effects of hydrophobic environment and pH by Aaron Kerman; V.S. Ananthanarayanan (pp. 1199-1210).

Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133–140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the μ-opioid receptor (‘TM2–3’) that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2–3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the μ-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2–3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix–helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2–3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2–3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.

Keywords: G protein-coupled receptor; Opioid receptor; Transmembrane helices; Conformational determinant; Detergent; Circular dichroism

Photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of siRNA by Sabrina Oliveira; Marjan M. Fretz; Anders Høgset; Gert Storm; Raymond M. Schiffelers (pp. 1211-1217).

Photochemical internalization (PCI) has been employed as a tool for site-specific intracellular delivery of a variety of molecules. In this study, for the first time, PCI has been employed to facilitate the endosomal escape of small interfering RNA (siRNA) molecules, which are the functional mediators of RNA interference (RNAi). In order to interact with the machinery that will induce post-transcriptional gene silencing, siRNA molecules need to enter the cytoplasm of the cells. This study shows that one of the important rate-limiting steps of siRNA silencing efficiency is the ability of siRNA molecules and/or complexes to escape from the endosomes into the cytosol of the cells. The target of this study, the epidermal growth factor receptor (EGFR), is known as an attractive target for cancer therapy. In this study, a 10-fold increased efficiency in knockdown of the EGFR protein was obtained when anti-EGFR siRNA treatment was combined with PCI as compared to siRNA treatment alone. The fact that this combined treatment resulted in a stronger silencing efficiency indicates that lower doses of siRNA can be used when PCI is employed to augment siRNA delivery. Lowering doses of siRNA would prevent saturation of the RNAi machinery and reduce off-target effects. In addition, local illumination of target tissue would only induce PCI in the desired cells, which can further increase the specificity of the treatment, supporting PCI as an attractive strategy to improve siRNA silencing efficiency.

Keywords: EGFR; Photochemical internalization; siRNA; Silencing efficiency; Cancer therapy

Quantum mechanical calculations of charge effects on gating the KcsA channel by Alisher M. Kariev; Vasiliy S. Znamenskiy; Michael E. Green (pp. 1218-1229).

A series of ab initio (density functional) calculations were carried out on side chains of a set of amino acids, plus water, from the (intracellular) gating region of the KcsA K⁺ channel. Their atomic coordinates, except hydrogen, are known from X-ray structures [D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, R. MacKinnon, The structure of the potassium channel: molecular basis of K⁺ conduction and selectivity, Science 280 (1998) 69–77; R. MacKinnon, S.L. Cohen, A. Kuo, A. Lee, B.T. Chait, Structural conservation in prokaryotic and eukaryotic potassium channels, Science 280 (1998) 106–109; Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T. Chait, R. MacKinnon, The open pore conformation of potassium channels. Nature 417 (2001) 523–526], as are the coordinates of some water oxygen atoms. The 1k4c structure is used for the starting coordinates. Quantum mechanical optimization, in spite of the starting configuration, places the atoms in positions much closer to the 1j95, more tightly closed, configuration. This state shows four water molecules forming a “basket” under the Q119 side chains, blocking the channel. When a hydrated K⁺ approaches this “basket”, the optimized system shows a strong set of hydrogen bonds with the K⁺ at defined positions, preventing further approach of the K⁺ to the basket. This optimized structure with hydrated K⁺ added shows an ice-like 12 molecule nanocrystal of water. If the water molecules exchange, unless they do it as a group, the channel will remain blocked. The “basket” itself appears to be very stable, although it is possible that the K⁺ with its hydrating water molecules may be more mobile, capable of withdrawing from the gate. It is also not surprising that water essentially freezes, or forms a kind of glue, in a nanometer space; this agrees with experimental results on a rather different, but similarly sized (nm dimensions) system [K.B. Jinesh, J.W.M. Frenken, Capillary condensation in atomic scale friction: how water acts like a glue, Phys. Rev. Lett. 96 (2006) 166103/1–4]. It also agrees qualitatively with simulations on channels [A. Anishkin, S. Sukharev, Water dynamics and dewetting transitions in the small mechanosensitive channel MscS, Biophys. J. 86 (2004) 2883–2895; O. Beckstein, M.S.P. Sansom, Liquid-vapor oscillations of water in hydrophobic nanopores, Proc. Natl Acad. Sci. U. S. A. 100 (2003) 7063–7068] and on featureless channel-like systems [J. Lu, M.E. Green, Simulation of water in a pore with charges: application to a gating mechanism for ion channels, Prog. Colloid Polym. Sci. 103 (1997) 121–129], in that it forms a boundary on water that is not obvious from the liquid state. The idea that a structure is stable, even if individual molecules exchange, is well known, for example from the hydration shell of ions. We show that when charges are added in the form of protons to the domains (one proton per domain), the optimized structure is open. No stable water hydrogen bonds hold it together; an opening of 11.0 Å appears, measured diagonally between non-neighboring domains as glutamine 119 carbonyl O–O distance. This is comparable to the opening in the MthK potassium channel structure that is generally agreed to be open. The appearance of the opening is in rather good agreement with that found by Perozo and coworkers. In contrast, in the uncharged structure this diagonal distance is 6.5 Å, and the water “basket” constricts the uncharged opening still further, with the ice-like structure that couples the K⁺ ion to the gating region freezing the entrance to the channel. Comparison with our earlier model for voltage gated channels suggests that a similar mechanism may apply in those channels.

Keywords: KcsA K; ⁺; channel; Gating; Proton; Ab initio calculation

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes by Elena A. Dutseva; Yuri N. Antonenko; Elena A. Kotova; Jochen R. Pfeifer; Ulrich Koert (pp. 1230-1237).

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β^5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β^6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.

Keywords: Abbreviations; BLM; bilayer lipid membrane; gA; gramicidin A; AlPcS; ₃; aluminum trisulfophthalocyanine; DPhPC; diphytanoylphosphatidylcholineChannel-forming peptide; Planar lipid bilayer; Ionic current; Sensitized photoinactivation; Membrane thickness; Hydrophobic mismatch

Structural and functional recovery of electropermeabilized skeletal muscle in-vivo after treatment with surfactant poloxamer 188 by John M. Collins; Florin Despa; Raphael C. Lee (pp. 1238-1246).

A critical requirement for cell survival after trauma is sealing of breaks in the cell membrane [M. Bier, S.M. Hammer, D.J. Canaday, R.C Lee, Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells, Bioelectromagnetics 20 (1999) 194–201; R.C. Lee, D.C. Gaylor, D. Bhatt, D.A. Israel, Role of cell membrane rupture in the pathogenesis of electrical trauma, J. Surg. Res. 44 (1988) 709–719; R.C. Lee, J.F. Burke, E.G. Cravalho (Eds.), Electrical Trauma: The Pathophysiology, Manifestations, and Clinical Management, Cambridge University Press, 1992; B.I. Tropea, R.C. Lee, Thermal injury kinetics in electrical trauma, J. Biomech. Engr. 114 (1992) 241–250; F. Despa, D.P. Orgill, J. Newalder, R.C Lee, The relative thermal stability of tissue macromolecules and cellular structure in burn injury, Burns 31 (2005) 568–577; T.A. Block, J.N. Aarsvold, K.L. Matthews II, R.A. Mintzer, L.P. River, M. Capelli-Schellpfeffer, R.L. Wollman, S. Tripathi, C.T. Chen, R.C. Lee, The 1995 Lindberg Award. Nonthermally mediated muscle injury and necrosis in electrical trauma, J. Burn Care and Rehabil. 16 (1995) 581–588; K. Miyake, P.L. McNeil, Mechanical injury and repair of cells, Crit. Care Med. 31 (2003) S496–S501; R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524–4528; J.D. Marks, C.Y. Pan, T. Bushell, W. Cromie, R.C. Lee, Amphiphilic, tri-block copolymers provide potent membrane-targeted neuroprotection, FASEB J. 15 (2001) 1107–1109; B. Greenebaum, K. Blossfield, J. Hannig, C.S. Carrillo, M.A. Beckett, R.R. Weichselbaum, R.C. Lee, Poloxamer 188 prevents acute necrosis of adult skeletal muscle cells following high-dose irradiation, Burns 30 (2004) 539–547; G. Serbest, J. Horwitz, K. Barbee, The effect of poloxamer-188 on neuronal cell recovery from mechanical injury, J. Neurotrauma 22 (2005) 119–132]. The triblock copolymer surfactant Poloxamer 188 (P188) is known to increase the cell survival after membrane electroporation [R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524–4528; Z. Ababneh, H. Beloeil, C.B. Berde, G. Gambarota, S.E. Maier, R.V. Mulkern, Biexponential parametrization of T2 and diffusion decay curves in a rat muscle edema model: Decay curve components and water compartments, Magn. Reson. Med. 54 (2005) 524–531]. Here, we use a rat hind-limb model of electroporation injury to determine if the intravenous administration of P188 improves the recovery of the muscle function. Rat hind-limbs received a sequence of either 0, 3, 6, 9, or 12 electrical current pulses (2 A, 4 ms duration, 10 s duty cycle). Magnetic resonance imaging (MRI) analysis, muscle water content and compound muscle action potential (CMAP) amplitudes were compared. Electroporation injury manifested edema formation and depression of the CMAP amplitudes. P188 (one bolus of 1 mg/ml of blood) was administrated 30 or 60 min after injury. Animals receiving P188 exhibited reduced tissue edema ( p<0.05) and increased CMAP amplitudes ( p<0.03). By comparison, treatment with 10 kDa neutral dextran, which produces similar serum osmotic effects as P188, had no effect on post-electroporation recovery. Noteworthy, the present results suggest that a single intravenous dose of P188 is effective to restore the structural integrity of damaged tissues with intact circulation.

Keywords: Abbreviations; ATP; adenosine triphosphate; BFM; biceps femoris muscles; CMAP; compound muscle action potential; CMC; critical micelle concentration; CPMG; Carr–Purcell–Meiboom–Gill; EDL; extensor digitorum longus; EMG; electromyography; H&E; hematoxylin and eosin; IP; intraperitoneal; IV; intravenous; KCl; potassium chloride; LR; lactated Ringers; MR; magnetic resonance; MRI; magnetic resonance imaging; P188; poloxamer 188; SaO2%; blood oxygen saturation; SOL; soleus; ST; slice thickness; T1; spin–lattice relaxation time; T2; spin–spin relaxation time; TR; repetition time; TE; echo timeElectroporation; Poloxamer 188; Skeletal muscle; Action potential; MRI

Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A₂ by scanning alanine mutagenesis by Lucimara Chioato; Elisangela Aparecida Aragão; Tatiana Lopes Ferreira; Alexandra Ivo de Medeiros; Lúcia Helena Faccioli; Richard J. Ward (pp. 1247-1257).

Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A₂ from the venom of Bothrops jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca²⁺-independent membrane damaging activity against liposome membranes were compared. Residues 116–119 and 122–125 in the C-terminal loop region are structural determinants for these activities, indicating that membrane permeabilization by the BthTx-I is an important general property in all the measured effects. The structural determinants of myotoxicity and myoblast membrane permeabilization are highly correlated, demonstrating that cultured C2C12 myoblasts are a good model for the myotoxic effect. However, comparison of the structural determinants for all activities revealed several differences in the structural determinants between the effects against myoblast and bacterial membranes, and further differences when compared to the liposome membrane damaging effect. These membrane dependent effects are interpreted to be the consequence of differences in the activation of the membrane bound form of the protein on biological and artificial membranes.

Keywords: Bothropstoxin-I; Myotoxin; Bactericide; Myoblast; Liposome; PLA2

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state by H. Raghuraman; Sandeep Shrivastava; Amitabha Chattopadhyay (pp. 1258-1267).

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.

Keywords: Abbreviations; 6-NBD-PC; 1-palmitoyl-2-(6-[; N; -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl)-; sn-; glycero-3-phosphocholine; 12-NBD-PC; 1-palmitoyl-2-(12-[; N; -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-; sn; -glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; MOPS; 3-(; N; -morpholino)propanesulfonic acid; NBD; 7-nitrobenz-2-oxa-1,3-diazol-4-yl; NBD-PE; N; -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine; NBD-cholesterol; 25-[-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; REES; red edge excitation shiftNBD lipids; DPPC; Phase transition; REES; Gel phase; NBD looping

Roughness of the plasma membrane as an independent morphological parameter to study RBCs: A quantitative atomic force microscopy investigation by M. Girasole; G. Pompeo; A. Cricenti; A. Congiu-Castellano; F. Andreola; A. Serafino; B.H. Frazer; G. Boumis; G. Amiconi (pp. 1268-1276).

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane–skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

Keywords: Surface roughness; Atomic force microscopy; Membrane–skeleton structure

Omiganan interaction with bacterial membranes and cell wall models. Assigning a biological role to saturation by Manuel Nuno Melo; Miguel A.R.B. Castanho (pp. 1277-1290).

Omiganan pentahydrochloride (ILRWPWWPWRRK-NH₂·5Cl) is an antimicrobial peptide currently in phase III clinical trials. This study aims to unravel the mechanism of action of this drug at the membrane level and address the eventual protective role of peptidoglycan in cell walls. The interaction of omiganan pentahydrochloride with bacterial and mammalian membrane models – large unilamellar vesicles of different POPC:POPG proportions – was characterized by UV-Vis fluorescence spectroscopy. The molar ratio partition constants obtained for the two anionic bacterial membrane models were very high ((18.9±1.3)×10³ and (43.5±8.7)×10³) and about one order of magnitude greater than for the neutral mammalian models ((3.7±0.4)×10³ for 100% POPC bilayers). At low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially for the most anionic systems. Membrane saturation can account for such observations and mathematical models were derived to further characterize the peptide–lipid interaction under those conditions; a possible relation between saturation and MIC was deduced; this was supported by differential quenching studies of peptide internalization. Interaction with the bacterial cell wall was assessed using Staphylococcus aureus peptidoglycan extracts as a model. A strong partition towards the peptidoglycan mesh was observed, but not as large as for the membrane models.

Keywords: Antimicrobial peptide; Saturation; Partition; Membrane; Peptidoglycan

Kinetics of pore formation by the Bacillus thuringiensis toxin Cry1Ac by Mélanie Fortier; Vincent Vachon; Lucie Marceau; Jean-Louis Schwartz; Raynald Laprade (pp. 1291-1298).

After binding to specific receptors, Cry toxins form pores in the midgut apical membrane of susceptible insects. The receptors could form part of the pore structure or simply catalyze pore formation and consequently be recycled. To discriminate between these possibilities, the kinetics of pore formation in brush border membrane vesicles isolated from Manduca sexta was studied with an osmotic swelling assay. Pore formation, as deduced from changes in membrane permeability induced by Cry1Ac during a 60-min incubation period, was strongly dose-dependent, but rapidly reached a maximum as toxin concentration was increased. Following exposure of the vesicles to the toxin, the osmotic swelling rate reached a maximum shortly after a delay period. Under these conditions, at relatively high toxin concentrations, the maximal osmotic swelling rate increased linearly with toxin concentration. When vesicles were incubated for a short time with the toxin and then rapidly cooled to prevent the formation of new pores before and during the osmotic swelling experiment, a plateau in the rate of pore formation was observed as toxin concentration was increased. Taken together, these results suggest that the receptors do not act as simple catalysts of pore formation, but remain associated with the pores once they are formed.

Keywords: Pore formation; Insecticidal toxin; Osmotic swelling assay; Bacillus thuringiensis; Manduca sexta

Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers by W. Xie; I. Kania-Korwel; P.M. Bummer; H.-J. Lehmler (pp. 1299-1308).

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) – the major component of pulmonary surfactant – using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC ( T_m) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on T_m and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS)>K(PFOA)>>K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds cause adverse biological effects.

Keywords: PFOS; PFOA; DSC; DPPC; DPH; TMA-DPH

Erratum to “Biophysics of sphingolipids I. Membrane properties of sphingosine, ceramides and other simple sphingolipids” [Biochim. Biophys. Acta 1758 (2006) 1902–1921] by Félix M. Goñi; Alicia Alonso (pp. 1309-1310).

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BBA - Biomembranes (v.1768, #5)