Lenka Beranova

Effect of heavy water on phospholipid membranes: experimental confirmation of molecular dynamics simulations

Although there were experimental indications that phospholipid bilayers hydrated with D(2)O express different biophysical properties compared with hydration by ordinary H(2)O, a molecular concept for this behavior difference was only recently proposed by a molecular dynamics simulations study [T. Róg et al., J. Phys. Chem. B, 2009, 113, 2378-2387]. Here we attempt to verify those theoretical predictions by fluorescence measurements on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Specifically, we determine the water isotope effect on headgroup hydration and mobility, lateral lipid diffusion and lipid backbone packing. Time-dependent fluorescence shift experiments show significantly slower dynamics and lower hydration of the headgroup region for a bilayer hydrated with D(2)O, an observation in good agreement with the calculated predicted differences in duration of lipid-lipid and lipid-water bridges and extent of water penetration into the bilayer, respectively. The water isotope effect on the lipid order parameter of the bilayer core (measured by fluorescence anisotropy) and lateral diffusion of lipid molecules (determined by two-focus fluorescence correlation spectroscopy) is close to the experimental errors of the experiments, however also refers to slightly more rigid organization of phospholipid bilayers in heavy water. This study confirms the view that the water isotope effect can be particularly found in time-resolved physicochemical properties of the membrane. Together with the simulations our experiments provide a comprehensive, molecular view on the effect of D(2)O on phospholipid bilayers.

Roscovitine sensitizes leukemia and lymphoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with selective antitumor activity. However, many primary tumors are TRAIL resistant. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. We show that roscovitine and TRAIL demonstrate synergistic cytotoxicity in hematologic malignant cell lines and primary cells. Pretreatment of TRAIL-resistant leukemia cells with roscovitine induced enhanced cleavage of death-inducing signaling complex-bound proximal caspases after exposure to TRAIL. We observed increased levels of both pro- and antiapoptotic BCL-2 proteins at the mitochondria following exposure to roscovitine. These results suggest that roscovitine induces priming of cancer cells for death by binding antiapoptotic BCL-2 proteins to proapoptotic BH3-only proteins at the mitochondria, thereby decreasing the threshold for diverse proapoptotic stimuli. We propose that the mitochondrial priming and enhanced processing of apical caspases represent major molecular mechanisms of roscovitine-induced sensitization to TRAIL in leukemia/lymphoma cells.

Principles and applications of fluorescence lifetime correlation spectroscopy

Two fluorescence spectroscopy concepts, fluorescence correlation spectroscopy and time correlated single photon counting (TCSPC) are employed in fluorescence lifetime correlation spectroscopy (FLCS) - a relatively new technique with several experimental benefits. In FLCS experiments, pulsed excitation is used and data are stored in a special time-tagged time-resolved mode. Mathematical treatment of TCSPC decay patterns of distinct fluorophores and their mixture enables to calculate autocorrelation functions of each of the fluorophores and thus their diffusion properties and concentrations can be determined separately. Moreover, crosscorrelation of the two signals can be performed and information on interaction of the species can be obtained. This technique is particularly helpful for distinguishing different states of the same fluorophore in different microenvironments. The first application of that concept represents the simultaneous determination of two-dimensional diffusion in planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers. The lifetime in both investigated systems differed because the lifetime of the dye is considerably quenched in the layer near the light-absorbing surface. This concept was also used in other applications: a) investigation of a conformational change of a labeled protein, b) detection of small amounts of labeled oligonucleotides bound to metal particles or c) elucidation of the compaction mechanism of different sized labeled DNA molecules. Moreover, it was demonstrated that FLCS can help to overcome some FCS experimental drawbacks.

Hydration and Mobility of Oxidized Phospholipid Bilayer: Fluorescence Solvent Relaxation and Fluorescence Correlation Spectroscopy Study

Oxidative stress often leads to truncation of the sn-2 chain of polyunsaturated phospholipids and introduction of aldehyde or carboxyl group at its end. Such oxidized phospholipids (OxPLs) change biophysical properties of a lipid bilayer, and are involved in pathogeneses of numerous diseases. Herein we investigate the influence of oxidation on the dynamics and hydration of model lipid membranes. Local mobility and hydration and lipid lateral diffusion were measured using fluorescence solvent relaxation and fluorescence correlation z-scan spectroscopy, respectively. Lipid mixtures of palmitoyloleoyl-phosphatidylcholine (POPC) and 10 mol% of one of four OxPL POPC analogues (with oleoyl chain truncated to: 5′-oxo-valeroyl, 9′-oxo-nonanoyl, glutaryl, or azelaoyl) were investigated. Atomic-scale molecular dynamics simulations were employed to give a rationale for the observed changes. Truncated tails of OxPLs were found to loop back toward the aqueous solution, which leads to increased membrane fluidity, i.e., both the local headgroup mobility and the lateral diffusion are increased. Moreover, these changes depend on the chemical nature of the oxidized chains and the salt present. Our results show that products of lipid oxidation may influence physiology not only by protein-mediated signaling, but also simply by altering physical properties of a lipid bilayer.