Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggeste

Characterization of sphingosine−phosphatidylcholine monolayers : effects of DNA

Monolayers of the naturally occurring cationic lipid sphingosine and its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area (p-A) isotherms and determined the monolayer dipole potential ¿ as a function of the mole fraction of sphingosine (XSph) with and without a charge-saturating concentration of DNA in the subphase. Both sphingosine and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents (XSph = 0.05) of sphingosine in a POPC monolayer condensed the film dramatically, by 20% at 20 mN/m. This effect is suggested to reflect a reorientation of the P--N+ dipole of the POPC headgroup (Säily, V. M. J.; Ryhänen, S. J.; Holopainen, J. M.; Borocci, S.; Mancini, G.; Kinnunen, P. K. J. Biophys. J. 2001, 81, 2135), in keeping with a simultaneous and pronounced increase in ¿. Mixed monolayers of sphingosine and POPC exhibited three critical mole fractions XSph of sphingosine, viz., 0.25, 0.6, and 0.83, at which the area/molecule reached a local minimum, followed by a pronounced expansion of the film. This suggests energetically favorable ordering, which allows the positively charged sphingosines to maximize their distance, so as to minimize the Coulombic repulsion. The presence of DNA affected the mixed POPC/sphingosine monolayers differently depending on the constituent lipid stoichiometry, yet the same discontinuities were evident as in the presence of DNA

Drug delivery by nanoparticles - facing the obstacles

There are numerous concepts of nanoparticle mediated drug delivery. The major advantage will be the option of targeted drug delivery to specific target cells thus avoiding high systemic loads of potentially toxic chemicals. Any kind of drug delivery by nanoparticles relies on delivery of the drug into the cell. In most cases that means drug delivery into the cytoplasm, and in some instances delivery of the drug to extracellular domains of transmembrane signalling molecules. Whenever viable cells are confronted with nanoparticles these are ingested by endocytosis rather then passage through the cell plasma membrane. Once inside endosomal vesicles the nanoparticles or at least their drug payload requires release into the cytoplasm in order to exert it’s biological effect. In order to monitor whether a drug delivered by nanoparticles is biologically active a toxic model drug, disulfiram, was chosen as a payload with micelle and liposome nanoparticles. L929 mouse fibroblasts were incubated with these disulfiram loaded naoparticles and cell viability was determined by quantification of celluar reductase activity. Applied nanoparticles are toxic to the cells. However, with respect to the disulfiram payload a 100-fold higher disulfiram concentration is required in comparison to free disulfiram for a biological effect. Hence, the toxic effect is most likely not due to the disulfiram delivered by the nanoparticles but rather to the amount of free disulfiram that is present in the nanoparticle preparation. Therefore it is advised to carefully characterize the nanoparticle suspension for the amount of free payload molecule

Morphology and Phase Behavior of Two-Component Lipid Membranes

The stability and shapes of domains with different bending rigidities in lipid membranes are investigated. These domains can be formed from the inclusion of an impurity in a lipid membrane or from the phase separation within the membrane. We show that, for weak line tensions, surface tensions and finite spontaneous curvatures, an equilibrium phase of protruding circular domains or striped domains may be obtained. We also predict a possible phase transition between the investigated morphologies