The prion fragment PrP106-127 adopts a secondary structure typical of aggregated fibrils in langmuir monolayers of brain lipid extract.

Understanding protein aggregation is essential to unveil molecular mechanisms associated with neurodegenerative diseases such as Alzheimer's, Huntington's and spongiform encephalopathy, particularly to determine the role of interaction with cell membranes. In this study, we employ Langmuir monolayers as cell membrane models to mimic interaction with the peptide KTNMHKHMAGAAAAGAVVGGLG-OH, a fragment from the human prion protein including residues 106-127, believed to be involved in protein aggregation. Using in situ polarization-modulated infrared reflection adsorption spectroscopy (PM-IRRAS) for Langmuir monolayers and FTIR for solid films, we found that PrP106-127 adopts mainly ß-sheets, random coils and ß-turns in Langmuir monolayers and in Langmuir-Blodgett (LB) and cast films. This also applies to monolayers and solid films made with PrP106-127 and a brain total lipid extract (BTLE). In contrast, some α-helices are observed in the secondary structure of PrP106-127 in monolayers, and especially in solid films, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In summary, in a model representing brain cells (BTLE), the secondary structure of PrP106-127 is typical of fiber aggregates, while aggregation is unlikely if PrP106-127 interacts with a membrane model (DOPC) characteristic of mammalian cells.

Challenges in Application of Langmuir Monolayer Studies To Determine the Mechanisms of Bactericidal Activity of Ruthenium Complexes.

The effects induced by antibiotics on the bacterial membrane may be correlated with their bactericidal activity, and such molecular-level interactions can be probed with Langmuir monolayers representing the cell membrane. In this study, we investigated the interaction between [Ru(mcbtz)2(PPh3)2] (RuBTZ, mcbtz = 2-mercaptobenzothiazoline) and [Ru(mctz)2(PPh3)2] (RuCTZ, mctz = 2-mercaptothiazoline) with Langmuir monolayers of a lipid extract of Escherichia coli, an extract of lipopolysaccharides (LPSs), and a zwitterionic phospholipid, dioleoylphosphatidyl choline (DOPC). RuBTZ and RuCTZ had little effects on DOPC, which is consistent with their negligible toxicity toward mammalian cells that may be approximated by a zwitterionic monolayer. Also little were their effects on LPSs. In contrast, RuBTZ and RuCTZ induced expansion in the surface pressure isotherms and decreased the compressional modulus of the E. coli lipid extract. While the more hydrophobic RuBTZ seemed to affect the hydrophobic tails of the E. coli extract monolayer to a larger extent, according to polarization modulation infrared reflection absorption spectroscopy results, evidence of a stronger RuBTZ interaction could not be confirmed unequivocally. Therefore, the interaction with the E. coli cell membrane cannot be directly correlated with the observed higher bactericidal activity of RuBTZ, in comparison to that of RuCTZ. This appears to be a case in which Langmuir monolayer studies do not suffice to determine the mechanisms responsible for the bactericidal activity.

Correlation of [RuCl3(dppb)(VPy)] cytotoxicity with its effects on the cell membranes: an investigation using Langmuir monolayers as membrane models.

One of the major challenges in drug design is to identify compounds with potential toxicity toward target cells, preferably with molecular-level understanding of their mode of action. In this study, the antitumor property of a ruthenium complex, mer-[RuCl3(dppb)(VPy)] (dppb = 1,4-bis(diphenylphosphine)butane and VPy = 4-vinylpyridine) (RuVPy), was analyzed. Results showed that this compound led to a mortality rate of 50% of HEp-2 cell with 120 ± 10 µmol L(-1), indicating its high toxicity. Then, to prove if its mode of action is associated with its interaction with cell membranes, Langmuir monolayers were used as a membrane model. RuVPy had a strong effect on the surface pressure isotherms, especially on the elastic properties of both the zwitterionic dipalmitoylphosphatidylcholine (DPPC) and the negatively charged dipalmitoylphosphatidylglycerol (DPPG) phospholipids. These data were confirmed by polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). In addition, interactions between the positive group from RuVPy and the phosphate group from the phospholipids were corroborated by density functional theory (DFT) calculations, allowing the determination of the Ru complex orientation at the air-water interface. Although possible contributions from receptors or other cell components cannot be discarded, the results reported here represent evidence for significant effects on the cell membranes which are probably associated with the high toxicity of RuVPy.

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells.

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.

Dibucaine effects on structural and elastic properties of lipid bilayers.

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.