melitten and stearic-acid

A crucial bottleneck in nonviral vector-mediated gene delivery is poor endosomal escape. Here, we constructed novel gene vectors by coupling the stearyl moiety to the N-terminus of the antimicrobial peptide melittin (stearyl-Mel) and its retro isomer (stearyl-rMel) due to their high membrane-lytic activity. As expected, stearyl-Mel showed obvious increases in endosome-lytic activity and transfection efficiency compared with the reported stearyl-TP10. More gratifyingly, the transfection efficiency of stearyl-rMel was around 10-fold greater than that of stearyl-Mel and almost reached the transfection levels of Lipofectamine 2000 due to the enhanced endosome-lytic activity. Furthermore, the stearyl-rMel/p53 plasmid complex exhibited higher p53 expression and antitumor activity than stearyl-Mel, confirming the fact that stearyl-rMel displayed higher transfection efficiency. Taken together, the combination of the stearyl moiety with retro melittin provides a novel framework for the development of excellent nonviral gene vectors.

Topics: Animals; Antimicrobial Cationic Peptides; Antineoplastic Agents; Cell Proliferation; Cells, Cultured; Chlorocebus aethiops; CHO Cells; COS Cells; Cricetulus; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Gene Transfer Techniques; Genetic Vectors; Humans; Melitten; Molecular Structure; Stearic Acids; Stereoisomerism; Structure-Activity Relationship; Transfection

Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phos

Topics: Amino Acid Sequence; Bee Venoms; Dimyristoylphosphatidylcholine; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Lipid Bilayers; Melitten; Molecular Sequence Data; Nitrogen Oxides; Phosphatidylglycerols; Phospholipids; Spin Labels; Stearic Acids; Temperature

Previous studies have shown that palmitic, stearic, oleic and linoleic acid levels in trophocytes prepared from the fat body of male Periplaneta americana are increased following treatment of the cells with hypertrehalosemic hormone (HTH). Melittin, an activator of phospholipase A2, mimicked the action of HTH by increasing the free fatty acid content in a concentration-dependent manner. The increase caused by HTH could be eliminated by pretreatment of the trophocytes with 1 mM 4'-bromophenacyl bromide (BPB), an inhibitor of phospholipase A2. BPB also decreases the concentration of free fatty acids in trophocytes not treated with HTH but by a smaller margin. Nordihydroguaiaretic acid (NDGA) and indomethacin, inhibitors of lipoxygenase and cyclooxygenase, respectively, eliminated the increase in free fatty acids evoked by HTH. In the absence of HTH both inhibitors increased the free fatty acid content of the trophocytes, an effect consistent with the known mode of action of these agents. None of the inhibitors tested, all of which blocked HTH activated trehalose synthesis, prevented activation of phosphorylase by HTH. This is taken as evidence that other downstream sites are also important in the regulation of trehalose production by the fat body. It is suggested that the increase in free fatty acids evoked by HTH, or metabolites of those fatty acids, may regulate the synthesis and release of trehalose from the trophocytes because of potential effects on trehalose phosphate synthase, trehalose 6-phosphate phosphatase, and the trehalose transport mechanism in the trophocyte membrane.

Topics: Acetophenones; Animals; Cyclooxygenase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Fat Body; Fatty Acids; Indomethacin; Insect Hormones; Linoleic Acid; Male; Masoprocol; Melitten; Neuropeptides; Oleic Acid; Palmitic Acid; Periplaneta; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Stearic Acids; Trehalose