Giovanna Zolese

Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines

The functions of N‐acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp‐214 in HSA or Trp‐212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp‐134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue. Proteins 2000;40:39–48.

A NEW FLUORESCENCE METHOD TO DETECT SINGLET OXYGEN INSIDE PHOSPHOLIPID MODEL MEMBRANES

A fluorescence method for detecting singlet oxygen (1O2) in model membranes is proposed. 1O2 was generated by hydrogen peroxide/sodium hypochlorite system. 1,3-Diphenylisobenzofuran (DPBF), a specific 1O2 trap, dissolved in organic solvents gives a strong fluorescence spectrum when excited at 410 nm. A similar spectrum, with a maximum at 455 nm, is obtained when DPBF is incorporated in unilamellar dipalmitoylphosphatidylcholine liposomes. The intensity of fluorescence spectrum decreases when DPBF-labeled liposomes are exposed to singlet oxygen. This decrease is sensitive to 1O2 traps and quenchers like tryptophan and sodium azide, to lipid membrane fluidity and to the concentration of sodium hypochlorite and hydrogen peroxide.

N-acylethanolamines as membrane topological stress compromising agents.

The effect of different N-acylethanolamines on the phase behaviour of fully hydrated egg phosphatidylethanolamines is reported. In particular, in the presence of N-acylethanolamines, the transition from the liquid-crystalline lamellar (L alpha) to the inverse hexagonal (HII) phase is observed at higher temperature with respect to the temperature transition of pure phosphatidylethanolamine. Moreover, in correspondence of this transition, an intermediate Q224 (space group Pn3m) cubic phase has been detected. Since the structure of this cubic phase presents unique topological analogies with the lipid bilayer organization, these data suggest the possible role of N-acylethanolamines in stabilizing the biological membranes by avoiding a sudden change to a non-bilayer phase in those tissues which undergo stress conditions.

Oleoylethanolamide Protects Human Sperm Cells from Oxidation Stress: Studies on Cases of Idiopathic Infertility

Abstract N-acylethanolamides are naturally occurring hydrophobic molecules usually present in a very small amount in many mammalian tissues and cells. The presence of N-acylethanolamides has also been demonstrated in human reproductive tracts and fluids, although their biological effects and molecular mechanisms of action are not yet completely elucidated. It is known that some N-acylethanolamides, such as oleoylethanolamide, have antioxidative properties. The aim of this study was to test whether oleoylethanolamide could protect sperm cells from reactive oxygen species-induced oxidative damage in cases of idiopathic infertility, because the excessive generation of these radicals was associated with this pathology. Our results show that 2.5 nM oleoylethanolamide in vitro supplementation significantly reduces DNA strand breaks both in fertile and infertile subjects. Moreover, oleoylethanolamide increases kinematic parameters, such as curvilinear velocity and amplitude of lateral head displacement and hyperactivation, both in the presence and in the absence of oxidative stress. Results of this study support the hypothesis of a possible protective action of oleoylethanolamide against reactive oxygen species, which could explain its beneficial effects on in vitro capacitated spermatozoa.

Effect of organotin compounds on membrane lipids: Fluorescence spectroscopy studies

The temperature dependence of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylamino-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) fluorescence anisotropy has been studied to investigate the influence of mono-n-butyltin chloride (MBTC), di-n-butyltin chloride (DBTC) and tri-n-phenyl chloride (TPTC) on the physicochemical state of dipalmitoyl phosphatidylcholine. Below the lipid chain melting transition (T m ), fluorescence anisotropy values of DPH and TMA-DPH are increased by the presence of the organotins, without important modifications of the phase transition temperature. A possible difference in localization of the organotin compounds is suggested by the differential effect of the probes. It is suggested that there is localization in the hydrophobic core of the bilayer for TPTC, and at the head-group level for DBTC, and a homogeneous distribution in the bilayer for MBTC. Similar studies have been performed in liposomal suspensions of cardiolipin, phosphatidylserine and egg phosphatidylcholine.

Plasma Membrane Perturbation Induced by Organotins on Erythrocytes from Salmo irideus Trout

Tributyltin chloride and its degradation products monobutyltin and dibutyltin act as water pollutants, owing to the use of tributyltin chloride as a biocide in marine paint formulations. These compounds are lipid-soluble and undergo bioaccumulation and bioconcentration. Salmo irideus trout erythrocytes were studied to evaluate the possible effects of these compounds on freshwater fish, which could be exposed to long-term effects due to bioaccumulation of organotins. Data showed that tributyltin increases the haemolysis rate, starting at 10 μM, while dibutyltin has a scant protective effect at each concentration tested. Similar studies were performed in the presence of carbon monoxide (CO), which is protective against membrane oxidative stress due to haemoglobin (Hb) auto-oxidation. In these conditions all the organotins tested induced an increase in the haemolysis rate. These results suggest that the consequence of auto-oxidation of Hb could condition the effects of some organotin compounds. Steady-state fluorescence of probes embedded in the lipidic part of the membrane was used to evaluate the modifications induced by organotins to the physico-chemical state of phospholipids.

LIPID-DRUG INTERACTION : THERMODYNAMIC AND STRUCTURAL EFFECTS OF ANTIMICOTIC FLUCONAZOLE ON DPPC LIPOSOMES

Abstract Calorimetry, X-ray diffraction, transmission electron microscopy and fluorescence techniques have been used to obtain thermodynamic and structural information on dipalmitoyl phosphatidyl choline (DPPC) liposomes doped by the drug fluconazole. The decrease of the gel to liquid crystalline phase transition temperature, as shown by DSC measurements, indicates that fluconazole imparts higher fluidity to the lipid matrix. X-ray diffraction and freeze fracture results show that the presence of the drug stabilizes the two-dimensional lamellar rippled phase Pβ′, prolonging its range of existence, till to room temperature. Fluorescence data show that fluconazole could increase the polarity of fluorescence probe microenvironment. Thus, all the data ensure that fluconazole affects the lipid membrane properties and support the hypothesis that antifungal fluconazole activity is related not only to its structural characteristics but also to its ability to interact with the lipid bilayer.

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.

Laurdan∗ fluorescence: a simple method to evaluate sperm plasma membrane alterations ☆

OBJECTIVE To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. DESIGN Pilot study. SETTING Andrology laboratory and biochemistry institute, medical school. PATIENT(S) Men whose semen was studied for infertility problems. INTERVENTIONS(S) No therapeutic intervention was performed on patients. MAIN OUTCOME MEASURE(S) Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. RESULT(S) Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 x 10(6) cells per milliliter, mean GP value was 0.303 +/- 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 +/- 0.026 (P<.001). These data indicate that the spermatozoa plasma membranes are characterized by liquid-crystalline phases with different ordering degree and polarity and that about 50% of samples with normal semen characteristics (> or =20 x 10(6) cells per milliliter) show high GP values. CONCLUSION(S) Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.

Effect of N-acylethanolamines with different acyl-chains on DPPC multilamellar liposomes

The influence of N-acylethanolamines with different acyl-chains on the physico-chemical state of neutral phospholipids was investigated using dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes. The thermal dependence of steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its charged derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was recorded. The N-acylethanolamines modified the DPPC phase transition temperature and broadened the transition temperature range in different ways depending on the N-acylethanolamines acyl chain characteristics. Our data suggest that the N-acylethanolamine acyl chain length and unsaturation play an important role in the interaction of these compounds with model membranes. The results show that long-chain-N-acylethanolamines interact largely with DPPC model membranes while a similar effect is not observed for the short ones.