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BBA - Biomembranes (v.1718, #1-2)

Editorial Board (pp. ii).

Behaviour of small solutes and large drugs in a lipid bilayer from computer simulations by D. Bemporad; C. Luttmann; J.W. Essex (pp. 1-21).

To reach their biological target, drugs have to cross cell membranes, and understanding passive membrane permeation is therefore crucial for rational drug design. Molecular dynamics simulations offer a powerful way of studying permeation at the single molecule level. Starting from a computer model proven to be able to reproduce the physical properties of a biological membrane, the behaviour of small solutes and large drugs in a lipid bilayer has been studied. Analysis of dihedral angles shows that a few nanosesconds are sufficient for the simulations to converge towards common values for those angles, even if the starting structures belong to different conformations. Results clearly show that, despite their difference in size, small solutes and large drugs tend to lie parallel to the bilayer normal and that, when moving from water solution into biomembranes, permeants lose degrees of freedom. This explains the experimental observation that partitioning and permeation are highly affected by entropic effects and are size-dependent. Tilted orientations, however, occur when they make possible the formation of hydrogen bonds. This helps to understand the reason why hydrogen bonding possibilities are an important parameter in cruder approaches which predict drug absorption after administration. Interestingly, hydration is found to occur even in the membrane core, which is usually considered an almost hydrophobic region. Simulations suggest the possibility for highly polar compounds like acetic acid to cross biological membranes while hydrated. These simulations prove useful for drug design in rationalising experimental observations and predicting solute behaviour in biomembranes.

Keywords: Molecular dynamics simulation; Constraint; β-blockers; DPPC membrane; Permeability

Characterization of cationic liposomes based on dimethyldioctadecylammonium and synthetic cord factor from M. tuberculosis (trehalose 6,6′-dibehenate)—A novel adjuvant inducing both strong CMI and antibody responses by Jesper Davidsen; Ida Rosenkrands; Dennis Christensen; Anil Vangala; Daniel Kirby; Yvonne Perrie; Else Marie Agger; Peter Andersen (pp. 22-31).

Incorporation of the glycolipid trehalose 6,6′-dibehenate (TDB) into cationic liposomes composed of the quaternary ammonium compound dimethyldioctadecylammonium (DDA) produce an adjuvant system which induces a powerful cell-mediated immune response and a strong antibody response, desirable for a high number of disease targets. We have used differential scanning calorimetry (DSC) to investigate the effect of TDB on the gel-fluid phase transition of DDA liposomes and to demonstrate that TDB is incorporated into DDA liposome bilayers. Transmission Electron Microscopy (TEM) and cryo-TEM confirmed that liposomes were formed when a lipid film of DDA containing small amounts of TDB was hydrated in an aqueous buffer solution at physiological pH. Furthermore, time development of particle size and zeta potential of DDA liposomes incorporating TDB during storage at 4 °C and 25 °C, indicates that TDB effectively stabilizes the DDA liposomes. Immunization of mice with the mycobacterial fusion protein Ag85B–ESAT-6 in DDA–TDB liposomes induced a strong, specific Th1 type immune response characterized by substantial production of the interferon-γ cytokine and high levels of IgG2b isotype antibodies. The lymphocyte subset releasing the interferon-γ was identified as CD4 T cells.

Keywords: Adjuvant; Immunomodulator; Liposome; Trehalose 6,6′-dibehenate; Dimethyldioctadecylammonium

Inhibition by calyculin A and okadaic acid of the Ca²⁺ release-activated Ca²⁺ entry pathway in rat basophilic leukemia cells: Evidence for regulation by Type 1/2A serine/threonine phosphatase activity by Nicholas E. Evans; Mark K.L. Forth; Anna K. Simpson; Michael J. Mason (pp. 32-43).

Using a combination of fluorescence measurements of intracellular Ca²⁺ ion concentration ([Ca²⁺]_i) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca²⁺ entry stimulated by activation of the Ca²⁺ release-activated Ca²⁺ (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca²⁺]_i to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca²⁺ rise associated with activation of CRAC uptake and inhibited Mn²⁺ entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca²⁺ stores is accompanied by a large depolarisation which is absolutely dependent upon Ca²⁺ entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.

Keywords: Phosphatase; CRAC; RBL cell; Calyculin A; Okadaic acid; Calcium

EPR study of lipid phase in renal cortical membrane organelles from intact and cadmium-intoxicated rats by Marta Žuvić-Butorac; Carol M. Herak-Kramberger; Dubravka Krilov; Ivan Sabolić; Janko N. Herak (pp. 44-52).

Numerous studies have demonstrated various structure/function correlations at the level of transport proteins in the kidney cell membranes and various intracellular organelles. However, characterization of the lipid phase of these membranes is rare. Here, we report the differences in lipid organization and dynamics of the brush-border membranes (BBM), basolateral membranes (BLM) and endocytotic vesicles (EV), isolated from the kidney cortex of intact rats, studied with the EPR spectroscopy of the spin-labeled membrane lipids. The EPR spectra were analyzed by comparing experimentally observed line shapes with the line shapes calculated according to the theoretical model developed for liquid crystals. In the fitting procedure, three different lipid domains were assumed, which revealed clear differences in the lipid ordering and rotational correlation times, as well as in the lipid partition of these domains in each of the three types of membranes. A similar approach, used to compare the spectroscopic characteristics of BBM from control and cadmium-intoxicated rats, showed significantly changed ordering and increased molecular mobility in the lipid phase of BBM from Cd-treated animals. As tested by an established fluorescence assay, the Cd-induced changes in the lipid mobility co localized with ∼5-fold higher conductance of BBM for potassium, with unchanged conductance for protons.

Keywords: Basolateral membrane; Brush border membrane; Endocytic vesicle; EPR spectroscopy; Heavy metal nephrotoxicity; Ion conductance; Membrane fluidity

Relationships of Cysteine and Lysine residues with the substrate binding site of the mitochondrial ornithine/citrulline carrier: An inhibition kinetic approach combined with the analysis of the homology structural model by Annamaria Tonazzi; Nicola Giangregorio; Ferdinando Palmieri; Cesare Indiveri (pp. 53-60).

To gain insights in the relationships of specific amino acid residues with the active site of the mitochondrial ornithine/citrulline carrier, we studied the effect of specific protein modifying reagents on the transport catalysed by the carrier reconstituted into liposomes. It was found that, besides the sulfhydryl reagents NEM, MTSEA, p-hydroxymercuribenzoate, diamide also the lysine reagents PLP, DIDS, SITS, the carboxyl reagents WRK, EDC and the arginine reagent methylglyoxal inhibited the carrier. NEM, MTSEA and PLP inhibited the ornithine/citrulline carrier with a completely competitive type of mechanism. A 1:1 interaction of NEM with the carrier molecule has been demonstrated. The results are in agreement with the localization of one sulfhydryl and at least one amino group in the substrate binding site. On the basis of the interferences between SH reagents and PLP in the transport inhibition, it has been deduced that the distance between the SH and the NH₂ residues of the active site should be comparable to the distance between the γ-NH₂ and COOH residues of the ornithine molecule. The structural model of the ornithine/citrulline carrier has been obtained by homology modelling using as template the ADP/ATP carrier structure. The combined analysis of the experimental data and the structural model allows to deduce that Cys-132 is located in the substrate binding site, flanked by at least one Lys residue.

Keywords: Abbreviations; p-OHMB; p-hydroxymercuribenzoate; MTSEA; (2-aminoethyl)methanethiosulfonate hydrobromide; NEM; N-ethylmaleimide; DTE; 1.4-dithioerythritol; PLP; pyridoxal 5-phosphate; SITS; 4-acetamido-4′-isothiocyanato-2,2′-stilbenedisulfonic acid; DIDS; 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid; WRK; Woodward's reagent K; EDC; 1-ethyl-3(3-dimethylaminopropyl)carbodiimmideMitochondria; Membrane; Transport; Carrier; Liposome; Ornithine; Structure; Reconstitution

Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains by Susanna Nybond; Y. Jenny E. Bj�rkqvist; Bodil Ramstedt; J. Peter Slotte (pp. 61-66).

The effects of ceramides with varying saturated N-linked acyl chains (C2–C14) on cholesterol displacement from sphingomyelin-rich domains and on the stability of ordered domains were studied. The bilayers examined were made from 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC),d- erythro-N-palmitoyl-sphingomyelin (PSM),d- erythro-N- acyl-sphingosine, and cholesterol (60:15:15:10 mol%, respectively). Cholestatrienol (CTL) ord- erythro-N- trans-parinoyl-sphingomyelin (tParSM) were used as reporter molecules (at 1 mol%) for the ordered domains, and 1-palmitoyl-2-stearoyl-(7-doxyl)- sn-glycero-3-phosphocholine (7SLPC) as a fluorescence quencher (30 mol%, replacing POPC) in the liquid-disordered phase. The results indicate that the ceramide had to have an N-linked acyl chain with at least 8 methylene units in order for it to displace cholesterol from the sphingomyelin-rich domains at the concentration used. The melting of the sphingomyelin-rich domain shifted to higher temperatures (compared to the ceramide-free control) with C2, C12 and longer chain ceramides, whereas C4–C10 ceramides led to domain melting at lower temperatures than control. This study shows that short-chain ceramides do not have the same effects on sterol- and sphingomyelin-rich domains as naturally occurring longer-chain ceramides have.

Keywords: Abbreviations; 7-SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; Cholestatrienol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; d; -; erythro-N; -palmitoyl-sphingomyelin; tParSM; d; -; erythro-N; -; trans; parinoyl-sphingosylphosphorylcholineSterol partitioning; Cholestatrienol; Trans-parinaric acid; Quenching; 7-doxyl phosphatidylcholine

Study of the interaction of sulfur dioxide derivative with cardiac sodium channel by Aifang Nie; Ziqiang Meng (pp. 67-73).

The effects of sulfur dioxide (SO₂) derivatives (bisulfite and sulfite, 1:3 M/M) on voltage-dependent sodium channel in isolated rat ventricular myocyte were studied using the whole cell patch-clamp technique. SO₂ derivatives increased sodium current ( I_Na) in a concentration-dependent manner. SO₂ derivatives at 10 μM significantly shifted steady-state inactivation curve of I_Na to more positive potentials, but did not affect the activation curve. SO₂ derivatives markedly shifted the curve of time-dependent recovery of I_Na from inactivation to the left, and accelerated the recovery of I_Na. SO₂ derivatives also significantly shortened the activation and inactivation time constants of I_Na. These results indicated that SO₂ derivatives produced concentration-dependent stimulation of cardiac sodium channels, which due mainly to the interaction of the drug with sodium channels in the inactivated state.

Keywords: Cardiomyocyte; Patch-clamp technique; Sodium channel; Sulfur dioxide

Biomembranes Cumulative Contents (pp. 74-74).

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