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Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated with such transport has been relatively tedious, even though significant progress has been made in the characterization of the intracellular trafficking of lipid/DNA complexes. Surprisingly little is known about the effects of these delivery vectors on cell functioning. In this report, we show that both cationic lipids and cationic lipid/DNA complexes mobilize the intracellular calcium. Removal of extracellular calcium did not significantly abolish this effect and preincubating cells with thapsigargin led to a decrease in [Ca2+]i, indicating that calcium was released mainly from internal calcium stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122, blocked the [Ca2+]i rise, suggesting an inositol dependent mechanism.

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.

The interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total reflection Fourier transform infra red (ATR-FTIR) spectroscopy. Related peptides have previously been shown to exhibit antibiotic and DNA transfection activities. The 26-residue LAH4X4 peptides were designed in such a manner to form amphipathic helical structures in membrane environments. Four histidines and four variable amino acids X constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. The dichroic ratio of ATR-FTIR spectra has been used to follow the pH-dependent transition from in-plane to transmembrane alignments upon increase in pH. A theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the Gibbs free energy of transition. The novel concept provides access to the free energy changes associated with the amino acids X incorporated into an extended alpha-helix and in the context of phospholipid bilayers. For the peptides of the series the Gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: LA approximately IS approximately FT approximately GV approximately WY.

This review describes the numerous and innovative methods used to study the structure and function of viral fusion peptides. The systems studied include both intact fusion proteins and synthetic peptides interacting with model membranes. The strategies and methods include dissecting the fusion process into intermediate stages, comparing the effects of sequence mutations, electrophysiological patch clamp methods, hydrophobic photolabelling, video microscopy of the redistribution of both aqueous and lipophilic fluorescent probes between cells, standard optical spectroscopy of peptides in solution (circular dichroism and fluorescence) and attenuated total reflection-Fourier transform infrared spectroscopy of peptides bound to planar bilayers. Although the goal of a detailed picture of the fusion pore has not been achieved for any of the intermediate stages, important properties useful for constraining the development of models are emerging. For example, the presence of alpha-helical structure in at least part of the fusion peptide is strongly correlated with activity; whereas, beta-structure tends to be less prevalent, associated with non-native experimental conditions, and more related to vesicle aggregation than fusion. The specific angle of insertion of the peptides into the membrane plane is also found to be an important characteristic for the fusion process. A shallow penetration, extending only to the central aliphatic core region, is likely responsible for the destabilization of the lipids required for coalescence of the apposing membranes and fusion. The functional role of the fusion peptides (which tend to be either nonpolar or aliphatic) is then to bind to and dehydrate the outer bilayers at a localized site; and thus reduce the energy barrier for the formation of highly curved, lipidic 'stalk' intermediates. In addition, the importance of the formation of specific, 'higher-order' fusion peptide complexes has also been shown. Recent crystallographic structures of core domains of two more fusion proteins (in addition to influenza haemagglutinin) has greatly facilitated the development of prototypic models of the fusion site. This latter effort will undoubtedly benefit from the insights and constraints gained from the studies of fusion peptides.

Several theories point out the correlation of the anaesthetic potenties of organic molecules with their membrane solubility. It has been shown that alkanols at nerve-blocking concentrations expand biological membranes (Seeman et al., 1969) or disturb order-parameters associated with the lipid (Chin & Goldstein, 1977). The mode of interaction of n-alkanols and lipids is unknown. In the present communication, the interaction of an homologous series of saturated aliphatic alkanols (C1-C13) with dipalmitoyl-dl-α-phosphatidylcholine (DPPC) is studied by a semi-empirical conformational analysis. This technique was developed to calculate the conformation of phospholipids (Brasseur et al., 1981) and other amphiphilic molecules (Brasseur et al., 1983) taking into account the anisotropic conditions of the interface. It takes into account not only the Van der Waals energies but also electrostatic and torsional energies (Brasseur et al., 1982). To simulate the lipid-water interface, the electrostatic energy i...

Adriamicin (ADM) is one of the most effective anthracycline glycoside antibiotic in the treatment of several types of cancer. Its clinical use is however limited by a specific cardiotoxicity. Deviation of electrons from NADPH supplemented cytochrome P-450 to the anthracycline results in the formation of a semiquinone radical which react with O2 to form toxic oxygen species (O2-, OH). These reactions were suggested to be involved in the cardiac toxicity process of ADM till the recent finding that cardiac microsomes do not participate to such reactions (Nohl & Jordan, 1983). Using the spin trapper DMPO combined with a flow technique, we were able to demonstrate that beef heart mitochondria, submitochondrial particles and complex I containing proteoliposomes catalyse the formation of O2- and OH species when incubated in presence of ADM and NADH. The semiquinone radical was also observed by ESR with a characteristic g value of 2.0024 and a line width of 0.4 mT. Interestingly, the 5-iminodaunorubicin analog did not induce the formation of oxygen radical species in good correlation with its weak cardiac toxicity. Membrane damages were further studied on heart mitochondria extracted from ADM-treated mice. A strong rigidifi-cation of the mitochondrial membrane was observed using fluorescence depolarization of a probe (DPH) embedded in the membrane. The rigidification can be considered as resulting from polymerization of peroxidized lipids in presence of O2- and OH species. No rigidification was observed on mitochondrial memmembrane extracted from 5-iminodaunorubicin-tretead mice in good agreement with our ESR results. © 1984 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.