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BBA - Biomembranes (v.1668, #1)

Editorial Board (pp. ii).

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism by Gregory Choi; Jianxin Guo; Alexandros Makriyannis (pp. 1-9).

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the β-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant α-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular³¹P–¹H and¹H–¹H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.

Keywords: Abbreviations; CD; circular dichroism; CNS; Central nervous system; DPC; Dodecylphosphocholine; SDS; sodium dodecyl sulfate; GPCRs; G-protein coupled receptors; MP-X; mastoparan-X; TM; transmembrane helixCannabinoid receptor; CB1; NMR; Helix 8; Circular dichroism; The fourth cytoplasmic loop

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver by C. Rothkopf; A. Fahr; G. Fricker; G.L. Scherphof; J.A.A.M. Kamps (pp. 10-16).

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [³H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.

Keywords: Phosphatidylserine; Liposome; Liver

Formation of supported phospholipid bilayers via co-adsorption with β-d-dodecyl maltoside by Hanna P. Vacklin; F. Tiberg; R.K. Thomas (pp. 17-24).

We have investigated the formation of supported model membranes via the adsorption of phospholipid–surfactant mixtures at the Si–water interface by specular neutron reflection. The adsorption of mixed micelles of the nonionic surfactant β-d-dodecyl maltoside and DOPC or POPC was determined as a function of bulk concentration, and using d₂₅-β-d-dodecyl maltoside, the composition of DOPC and POPC bilayers was determined. Bilayer thicknesses of 39±3 Å for DOPC and 41±3 Å for POPC agree well with data from bulk lamellar phases for both lipids, and the average area per lipid molecule can be varied from 62 to 115 Å² by varying the bulk concentrations used. The amount of surfactant in the bilayer is very sensitive to the bulk volume-to-surface area ratio, but it can be fully eliminated by ensuring a sufficiently large dilution/rinsing volume of the solution.

Keywords: Phospholipid bilayer; DOPC; Dodecyl maltoside; Micelle; Adsorption; Neutron reflection

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers by Masataka Kusube; Hitoshi Matsuki; Shoji Kaneshina (pp. 25-32).

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl- N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl- N, N-dimethylethanolamine (DPMe₂PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L_β) or ripple gel (P_β′) phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature–pressure boundary, d T/d p, was in the range of 0.220–0.264 K MPa⁻¹ depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L_c) phase to the lamellar gel (L_β or L_β′) phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L_c/L_β transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L_c/L_α transition was observed at a temperature higher than the main-transition temperature. The main (L_β/L_α) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L_β′, P_β′ and pressure-induced interdigitated gel (L_βI) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe₂PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.

Keywords: Bilayer membrane; N; -methylated phosphatidylethanolamine; Phase transition; Phospholipid; Pressure; Vesicle

Permeability changes of connexin32 hemi channels reconstituted in liposomes induced by extremely low frequency, low amplitude magnetic fields by Alfonsina Ramundo-Orlando; Annalucia Serafino; Rosangela Schiavo; Micaela Liberti; Guglielmo d'Inzeo (pp. 33-40).

The effect of extremely low frequency and low amplitude magnetic fields on gap junctional permeability was investigated by using reconstituted connexin32 hemi channel in liposomes. Cytochrome c was loaded inside these proteoliposomes and its reduction upon addition of ascorbate in the bulk aqueous phase was adopted as the index of hemi channel permeability. The permeability rate of the hemi channels, expressed as Δ A/min, was dependent on the incubation temperature of proteoliposomes. The effect of exposures to magnetic fields at different frequencies (7, 13 and 18 Hz) and amplitudes (50, 50 and 70 μT, respectively), and at different temperatures (16, 18 and 24 °C) was studied. Only the exposure of proteoliposomes to 18-Hz ( B_acpeak and B_dc=70 μT) magnetic field for 60 min at 16±0.4 °C resulted in a significant enhancement of the hemi channel permeability from Δ A/min=0.0007±0.0002 to Δ A/min=0.0010±0.0001 ( P=0.030). This enhancement was not found for magnetic field exposures of liposomes kept at the higher temperatures tested. Temperature appears to influence lipid bilayer arrangement in such a way as being capable to mask possible effects induced by the magnetic field. Although the observed effect was very low, it seems to confirm the applicability of our model previously proposed for the interaction of low frequency electromagnetic fields with lipid membrane.

Keywords: Gap junction; Connexin32 hemi channel; Channel reconstitution; Permeability change; Liposome; Magnetic field

c-Fos and phosphatidylinositol-4,5-bisphosphate reciprocally reorganize in mixed monolayers by Graciela A. Borioli; Beatriz L. Caputto; Bruno Maggio (pp. 41-52).

The transcription factor c-Fos has surface thermodynamic properties that allow it to differentially interact with phospholipids, especially PIP₂. It regulates phospholipid metabolism both in vivo and in vitro, and modulates degradation of phospholipid monolayers by phospholipases in a way that depends on the membrane intermolecular packing (i.e., surface lateral pressure). With the aim to understand details of the interactions of c-Fos at the membrane level, we studied the surface packing, dipole potential, compressibility and topography of mixed films of the protein with PIP₂. We show that c-Fos changes the packing of liquid-expanded PIP₂ monolayers, in a different manner with respect to its effect on the similarly liquid-expanded dilauroylphosphatidylcholine monolayers. The changes at the local molecular level are transduced to long-range inhomogeneities of the surface, detected by Brewster angle (BAM) and epifluorescence microscopy (EFM). Our results highlight the capacity of c-Fos to alter the packing and dipole potential of the lipid–protein interface. This involves variations of the surface in-plane elasticity and lateral segregation of phase domains. These dynamic, reversible alterations of surface organization provide a basis by which c-Fos may transduce molecular information at the membrane level.

Keywords: Abbreviations; π; surface lateral pressure; MARCKS; myristoylated alanine-rich C kinase substrate; GAP-43; growth-associated protein; CAP-23; cortical cytoskeleton-associated protein; PIP; ₂; phosphatidylinositol-4,5-bisphosphate; DAG; diacylglycerol; NBD-PE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine-; N; -(7-nitro-2-1,3-benzoxadiazol-4-yl); PLA; ₂; phospholipase A; ₂; PLC; phospholipase C; PC; dilauroylphosphatidylcholine; EFM; epifluorescence microscopy; BAM; Brewster angle microscopyLipid–protein interaction; PIP; ₂; Brewster angle microscopy; Epifluorescence microscopy; Surface topography

Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition by Csilla �zvegy-Laczka; Gabriella K�bl�s; Bal�zs Sarkadi; Andr�s V�radi (pp. 53-63).

The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

Keywords: Abbreviations; ABC; ATP binding cassette; ABCP; placenta specific ABC transporter; BCRP; breast cancer resistance protein; β-gal.; β-galactosidase; Hst; Hoechst 33342; MDR1; human multidrug resistance protein (P-glycoprotein, ABCB1); MRP; human multidrug resistance associated protein, ABCC1; MXR; mitoxantrone resistance protein; MTX; methotrexate; MX; mitoxantrone; R123; rhodamine 123; Sf9 cells; Spodoptera frugiperda; ovarian cells; SN-38; 7-ethyl-10-hydroxy-camptothecin; TM; transmembrane; wt; wild-typeMultidrug half-transporter; Single amino acid mutant; Cell surface localization; Membrane ATPase; Vesicular transport; Fluorescent dye extrusion

Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle by J.A. Fr�as; J.A. Cadefau; C. Prats; M. Mor�n; A. Meg�as; R. Cuss� (pp. 64-74).

Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose gradients (27–32–34–38–45%, wt/wt). All the fractions were characterized in terms of calcium content, Ca²⁺/Mg²⁺-ATPase activity, and radioligand binding of [³H]-PN 200-110 and [³H]ryanodine, specific to dihydropyridine-sensitive calcium channels and ryanodine receptors, respectively. Gradient fractions of muscles stimulated for 24 h underwent acute changes in the pattern of protein bands. First, light fractions from longitudinal sarcoplasmic reticulum, enriched in Ca²⁺-ATPase activity, R₁ and R₂, were greatly reduced (67% and 51%, respectively); this reduction was reflected in protein yield of crude microsomal fractions prior to gradient loading (25%). Second, heavy fractions from the sarcoplasmic reticulum were modified, and part (52%) of the R₃ fraction was shifted to the R₄ fraction, which appeared as a thick, clotted band. Quantification of [³H]-PN 200-110 and [³H]-ryanodine binding revealed co-migration of terminal cisternae and t-tubules from R₃ to R₄, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may be part of a physiological response to fatigue.

Keywords: Muscle electrostimulation; Sarcoplasmic reticulum; Triad; Calcium accumulation

The Folch–Lees proteolipid induces phase coexistence and transverse reorganization of lateral domains in myelin monolayers by C.M. Rosetti; R.G. Oliveira; B. Maggio (pp. 75-86).

Solvent solubilized myelin membranes spread as monomolecular layers at the air–water interface show a heterogeneous pattern at all surface pressures. In order to asses the role of myelin protein and lipid components in the surface structuring we compared the topography, as seen by Brewster angle microscopy (BAM) and epifluorescence microscopy, of monolayers made from mixtures containing all myelin lipids (except gangliosides) and variable proportions of Folch–Lees proteolipid protein (PLP, the major protein component of myelin). The presence of the single PLP, in the absence of the other myelin proteins, can reproduce the surface pattern of the whole myelin extract films in a concentration-dependant manner. Moreover, a threshold mole fraction of PLP is necessary to induce the lipid–protein component reorganization leading to the appearance of a rigid (gray) phase, acting as a surface skeleton, at low surface pressures and of fractal clusters at high surface pressures. The average size of those clusters is also dependent on the PLP content in the monolayer and on the time elapsed from the moment of film spreading, as they apparently result from an irreversible lateral aggregation process. The transverse rearrangement of the monolayer occurring under compression was different in films with the highest and lowest PLP mole fractions tested.

Keywords: Monolayer; Lipid–protein interface; Phase coexistence; Folch–Lees proteolipid; Epifluorescence microscopy; Brewster angle microscopy

Study of structure and orientation of mesentericin Y105, a bacteriocin from Gram-positive Leuconostoc mesenteroides, and its Trp-substituted analogues in phospholipid environments by Sabine Castano; Bernard Desbat; Antoine Delfour; Jean Marie Dumas; Alexandra da Silva; Jean Dufourcq (pp. 87-98).

Mesentericin Y105 (Mes-Y105) is a bacteriocin secreted by Leuconostoc mesenteroides which is particularly active on Listeria. It is constituted by 37 residues and reticulated by one disulfide bridge. It has two W residues, W18 and W37, which can be studied by fluorescence. Two single substituted W/F analogues were synthesized (Mes-Y105/W18 and Mes-Y105/W37) to differentiate the local environment around each W and to study their changes in the presence of lipid vesicles.Fluorescence experiments show that, for the pure Trp-analogues, W18 and W37 are fully exposed to solvent whatever pH and buffer conditions. In the presence of lipid vesicles, both became buried. Lipid affinities were estimated: they are weak for zwitterionic phospholipids but an order of magnitude higher for negatively charged phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. On negatively charged PG lipids, Mes-Y105 and Mes-Y105/W37 display comparable lipid affinities. A decrease in lipid affinity is observed for Mes-Y105/W18 compared to Mes-Y105, which means that W37 would seem to be required for increased lipid selectivity. In the lipid-bound state W18 is strongly dehydrated, probably embedded into the acyl chains, while W37 stands more at the interface.Mes-Y105 was also studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS), alone and in various phospholipid environments, to obtain structural information and to assess lipid perturbations. At nanomolar concentrations close to those required for anti-Listeria activity, Mes-Y105 forms films at the air/water interface and inserts into negatively charged lipid monolayers. In situ infrared data show that Mes-Y105 binding only affects the polar head group vibrations while the lipid order of the acyl chains remains unaffected. The PMIRRAS show that Mes-Y105 folds into an N-terminal antiparallel β-sheet followed by an α-helix, both structures being tilted (40°) compared to the normal at the interface, which is in agreement with the thickness estimated by Brewster angle microscopy (BAM). All these data support the proposal of a new model for Mes-Y105 at the membrane interface.

Keywords: Abbreviations; Mes-Y105; mesentericin; POPC; palmitoyl-oleyl-phosphatidylcholine; PS; phosphatidylserine; DMPC; dimyristoyl-phosphatidylcholine; EPC; egg phosphatidylcholine; DMPG; dimyristoyl-phosphatidylglycerol; DPPG; dipalmitoyl-phosphatidylglycerol; TFE; trifluoroethanol; PMIRRAS; polarization modulation infrared reflection absorption spectroscopy; BAM; Brewster angle microspopy; CD; circular dichro�sm; FRET; fluorescence resonance energy transferMesentericin; Bacteriocin; Lipid-bound structure; Peptide orientation; Fluorescence; PMIRRAS

New method for determining tie-lines in coexisting membrane phases using spin-label ESR by Yun-Wei Chiang; Jiang Zhao; Jing Wu; Yuhei Shimoyama; Jack H. Freed; Gerald W. Feigenson (pp. 99-105).

A full description of coexisting phases includes their respective compositions, which are provided by the thermodynamic tie-lines. Fluorescence microscopy enables visualization of coexisting lipid phases in giant unilamellar phases, but the composition information is missing. For cholesterol-containing lipid mixtures, knowledge of the compositions of the coexisting phases is important for understanding the nature of “membrane rafts�?. We propose and demonstrate a new method, based on ESR spectroscopy, for determining tie-lines in regions of two-phase coexistence in a ternary lipid mixture. Over 100 different lipid compositions containing the spin-labeled phospholipid 16-PC in or near the two-phase coexistence region of the liquid-disordered and the gel phases of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) were studied to determine five tie-lines, spread over virtually the full range of this coexistence region. The method is based on the facts that (1) along a tie-line the ESR spectrum must be a superposition of the two ESR spectra from the respective single phases at the phase boundaries (connected by the tie-line) in a ratio given by the lever rule; (2) along a tie-line the partition coefficient, K_p, for the spin-label, which is also determined in our method, must be constant. We do find that K_p for 16-PC is close to unity, but its value depends on the particular tie-line. The coexisting phases in equilibrium are characterized by the K_p of the spin-label and its respective dynamic parameters obtained from fitting the ESR spectra to dynamical models.

Keywords: Tie-line; Partition coefficient; Membrane phase coexistence; ESR; Ternary lipid mixture

A model of sodium channels by Taira Vora; Ben Corry; Shin-Ho Chung (pp. 106-116).

We have explored the permeation and blockage of ions in sodium channels, relating the channel structure to function using electrostatic profiles and Brownian dynamics simulations. The model used resembles the KcsA potassium channel with an added external vestibule and a shorter selectivity filter. The electrostatic energy landscape seen by permeating ions is determined by solving Poisson's equation. The two charged amino acid rings of Glu-Glu-Asp-Asp (EEDD) and Asp-Glu-Lys-Ala (DEKA) around the selectivity filter region are seen to play a crucial role in making the channel sodium selective, and strongly binding calcium ions such that they block the channel. Our model closely reproduces a range of experimental data including the current–voltage curves, current–concentration curves and blockage of monovalent ions by divalent ions.

Keywords: Sodium channel; Permeation; Ion channel; Brownian dynamics; Electrostatics; Blockage; Selectivity

Formation of homogeneous unilamellar liposomes from an interdigitated matrix by Alla Polozova; Xingong Li; Tong Shangguan; Paul Meers; Daniel R. Schuette; Nozomi Ando; Sol M. Gruner; Walter R. Perkins (pp. 117-125).

Phospholipid–ethanol–aqueous mixtures containing bilayer-forming lipids and 20–50 wt.% of water form viscous gels. Further hydration of these gels results in the formation of liposomes whose morphology depends upon the lipid type. Upon hydration of gels containing mixtures of the lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), small homogeneous and unilamellar liposomes were produced. In contrast, hydration of gels containing only POPC resulted in formation of large multilamellar liposomes. Likewise, mulitlamellar liposomes resulted when this method was applied to form highly fusogenic liposomes comprised of the novel negatively charged N-acyl-phosphatidylethanolamine (NAPE) mixed with di-oleoyl-phosphatidylcholine (DOPC) (7:3) [T. Shangguan, C.C. Pak, S. Ali, A.S. Janoff, P. Meers, Cation-dependent fusogenicity of an N-acyl phosphatidylethanolamine, Biochim. Biophys. Acta 1368 (1998) 171–183]. In all cases, the measured aqueous entrapment efficiencies were relatively high. To better understand how the molecular organization of these various gels affects liposome morphology, we examined samples by freeze-fracture transmission electron microscopy and X-ray diffraction. We found that phospholipid–ethanol–water gels are comprised of highly organized stacks of lamellae. A distinct feature of the gel samples that result in small unilamellar liposomes is the combination of acyl chain interdigitation and net electrostatic charge. We speculate that the mechanism of unilamellar liposome formation proceeds via formation of stalk contacts between neighboring layers similar to membrane hemifusion intermediates, and the high aqueous entrapment efficiencies make this liposome formation process attractive for use in drug delivery applications.

Keywords: Abbreviations; POPC; palmitoyl-oleoyl-phosphatidylcholine; POPG; palmitoyl-oleoyl-phosphatidylglycerol; DOPC; di-oleoyl-phosphatidylcholine; NBD-PE; nitrobenzoxadiazol-phoshatydylethanolamine; NAPE; N; -acyl-phosphatidylethanolamine; DPPC; di-palmitoyl-phoshatidylcholine; DSPC; di-stearoyl-phosphatidylcholine; SUV; small unilamellar vesiclesHomogeneous; Liposome; Matrix

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component by Nawel Mahrour; Roxana Pologea-Moraru; Mihaela G. Moisescu; St�phane Orlowski; Philippe Lev�que; Lluis M. Mir (pp. 126-137).

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 μs) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed (≈1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells—endocytosis.

Keywords: Abbreviations; EMF; electromagnetic field; LY; Lucifer Yellow; FD; FITC-Dextran; GSM; Global System for Mobile communications; CW; continuous wave; SAR; specific absorption rateFluid-phase endocytosis; Electromagnetic field; Electric field

A structural study of the myristoylated N-terminus of ARF1 by Thad A. Harroun; Jeremy P. Bradshaw; Kia Balali-Mood; John Katsaras (pp. 138-144).

The effect of myristoylation on the 15-amino-acid peptide from the membrane-binding N-terminus of ADP ribosylation factor 1 (ARF1) was studied using neutron diffraction and circular dichroism. A previous study on the non-acylated form indicated that the peptide lies parallel to the membrane, at a shallow depth and in the vicinity of the phosphorylcholine headgroups. It was suggested that the helix does not extend past residue 12, an important consequence for the linking region of the ARF1 protein. In this paper, we show that the result of myristoylation is to increase the helical content reaching the peptide's C-terminus, resulting in the formation of a new hydrophobic face. This increased helicity may augment the entire protein's membrane-binding affinity, indicating that ARF1 effectively has two interdependent membrane-binding motifs.

Keywords: ADP ribosylation factor (ARF); Phospholipid; Neutron diffraction; Myristoylation

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide by Kenji Fukuzawa; Yasuaki Saitoh; Kaori Akai; Kentaro Kogure; Satoru Ueno; Akira Tokumura; Masaki Otagiri; Akira Shibata (pp. 145-155).

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.

Keywords: Abbreviations; BSA; bovine serum albumin; aBSA; acetylated BSA; mBSA; methylated BSA; nBSA; native BSA; DCP; dicetylphosphate; EDTA; ethylenediamine tetraacetic acid; EGTA; bis(2-aminoethyl ether)ethyleneglycol tetraacetic acid; EYPC; egg yolk phosphatidylcholine; HEPES; 2-hydroxyethylpiperazine-2-ethanesulfonic acid; NTA; nitrilotriacetic acid (nitrilotriacetate); PC-OOH; hydroperoxides of egg yolk phosphatidylcholine; poly-Glu; poly-; l; -glutamic acid; poly-Lys; poly-; l-; lysine; poly (Lys-Phe); poly-; l-; lysine-; l-; phenylalanine; SA; stearylamine; TBA; thiobarbituric acid; X; xanthine; XO; xanthine oxidaseSerum albumin; Lipid peroxidation; Superoxide; Liposome; Membrane; Antioxidant; Free radical; Xanthine oxidase; Nitrilotriacetate; Iron

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BBA - Biomembranes (v.1668, #1)