Biomedical subjects

M Teresa Lamy

Publications and source records attributed to M Teresa Lamy.

6 recordsLinked to original sources

PubMed2006

Low cholesterol solubility in DODAB liposomes.

Through the analysis of the ESR spectra of spin labels, we investigated the thermotropic properties of dioctadecyl dimethylammonium bromide (DODAB) liposomes, in low and high ionic strength, with different cholesterol contents. The cationic lipid gel phase is stabilized by the presence of ions, the bilayer having a higher gel/fluid transition temperature (Tm) in high ionic strength. As found for low ionic strength [Benatti, C.R., Feitosa, E., Fernandez, R.M., Lamy-Freund, M.T., 2001. Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels. Chem. Phys. Lipids, 111, 93-104], high salt DODAB membranes also present a clear coexistence of the two phases around Tm. Cholesterol solubility in DODAB bilayers seems to be rather low, as the coexistence of DODAB and cholesterol-rich domains can be clearly detected by spin labels, for cholesterol concentration as low as 15 mol% of the total lipid. For lower cholesterol concentrations, the effect of cholesterol in DODAB bilayers is similar to that in phospholipids. For concentrations at or above 45 mol% of cholesterol, spin labels do not detect the coexistence of structurally different domains.

Cholesterol↗

PubMed2006

Laurdan in fluid bilayers: position and structural sensitivity.

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41 degrees C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel-fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.

Journal Article↗

PubMed2005

Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion.

Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe.

Cyclic N-Oxides↗

PubMed2005

Peptide-lipid interaction monitored by spin labeled biologically active melanocortin peptides.

The present work comparatively analyzes the interaction of alpha-MSH and its more potent and long-acting analog [Nle4, D-Phe7]alpha-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pKa 7.5, possibly that of His6, can be clearly monitored by peptide-lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.

Electron Spin Resonance Spectroscopy↗

PubMed2004

Structural characterization of diC14-amidine, a pH-sensitive cationic lipid used for transfection.

The structure of N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC(14)-amidine) cationic vesicles, used for transfection, was investigated at different pH values and ionic strengths, through the analysis of the electron spin resonance (ESR) spectra of spin labels. Phospholipid derivatives, spin labeled at the 5th and 16th C-atoms along the hydrocarbon chain, incorporated in diC(14)-amidine bilayers, show that the bilayer structure is highly sensitive to the pH value of the medium, due to the two titratable groups present in the amphiphile. Compared with samples at higher pH values, the double charged diC(14)-amidine at pH 3 presents a rather non-organized bilayer gel phase, and a much lower gel-fluid temperature transition, in accord with a strong headgroup electrostatic repulsion. In addition, the structure was found to be highly dependent on the ionic strength of the medium. However, pH 3 diC(14)-amidine bilayer, in the fluid phase, was found to be slightly more closely packed than those at pH 7.4 or 9.0, which are less charged. Parallel to that, the larger isotropic hyperfine splitting measured for nitroxides in the center of the pH 3 diC(14)-amidine bilayer suggests a higher membrane polarity for the highly charged low pH sample.

Amidines↗

PubMed2003

Structural study of melanocortin peptides by fluorescence spectroscopy: identification of beta-(2-naphthyl)-D-alanine as a fluorescent probe.

Several cyclic disulfide alpha-melanocyte stimulating hormone (alpha-MSH) analogues containing the aromatic fluorescent amino acid beta-(2-naphthyl)-D-alanine (D-Nal) have high affinity and selectivity for the melanocortin (MC)-4 receptor. Considering the possible relevant role played by the lipid phase in the peptide-receptor interaction, the structures of two cyclic alpha-MSH analogues, containing both Trp and D-Nal fluorophores, were investigated by steady-state and time-resolved fluorescence spectroscopy, in aqueous solution and in the presence of dimyristoyl phosphatidylglycerol (DMPG) vesicles, and compared with that of the natural peptide. The amino acid D-Nal gives a unique de-excitation fluorescence profile, with an excited state lifetime much longer than those of Trp, allowing good distinction between the two fluorophores. The cyclic analogues' aqueous structures seem to be adequate for membrane penetration, as Trp fluorescence indicates that, in both aqueous and lipid media, the Trp environment in the cyclic peptides is similar to that of alpha-MSH when incorporated in lipid bilayers. Trp, in the cyclic analogues, seems to penetrate deeper in the bilayer than in the native peptide. The amino acid D-Nal was also found to penetrate deep into the lipid bilayer, having its excited-state lifetime drastically changed from aqueous solution to lipid medium. The present work shows that D-Nal may serve as a fluorescent probe for studies of MC peptides and suggests that the high affinity and selectivity of the cyclic peptides to the MC4 membrane receptor could be related to their deeper penetration into the bilayer core.

Alanine↗