PubMed HealthSearch

Biomedical subjects

G Cervato

Publications and source records attributed to G Cervato.

15 recordsLinked to original sources

Calcitonin-induced changes in the organization of sulfatide-containing membranes.

The interactions of salmon calcitonin with glycosphingolipid sulfatide are studied by right angle light scattering from the lipid suspension, by the excimer to monomer ratio (E/M) of the fluorescence intensity of pyrene phosphatidylcholine and pyrene sulfatide and by the leakage of carboxyfluorescein. It was found that calcitonin strongly modified the structure of the sulfatide aggregate, as indicated by the light scattering determinations. At a lipid peptide ratio 100:1 (molar ratio) light scattering from the suspension was negligible, indicating the formation of peptide-sulfatide complexes with a structure different from that of the lipid aggregate. The interactions of calcitonin with sulfatide when the latter is a component of a bilayer were also evaluated. A specific calcitonin-membrane sulfatide interaction was demonstrated by determining the temperature-dependent E/M of pyrene phosphatidylcholine and pyrene sulfatide in dipalmitoyl phosphatidylcholine/sulfatide (80:20, molar ratio) liposomes. The E/M curves were modified by calcitonin only when the liposomes were labelled with fluorescent sulfatide which probes the sulfatide behavior in the membrane. Furthermore, the addition of calcitonin to the incubation medium of liposomes containing sulfatide promoted the release of vesicle entrapped carboxyfluorescein without disrupting the bilayer structure, the release being correlated with the amount of sulfatide in the bilayer and the calcitonin concentration in the medium.

1,2-Dipalmitoylphosphatidylcholine

Pyrene derivatives as markers of transbilayer effect of lipid peroxidation on neuronal membranes.

Two different pyrene derivatives, namely 12-(1-pyrene)dodecanoic acid (P12-FA) and N-(12-(1-pyrene)dodecanoyl)-galactosylsphingosine I3-sulfate (P12-CS) have been used to follow lipid peroxidation both in model and natural membranes. The malondialdehyde (MDA) production in small unilamellar vesicles of dipalmitoylphosphatidylcholine/arachidonic acid (80:20, molar ratio), symmetrically labelled with both probes determined a progressive decrease of pyrene fluorescence due to an involvement of pyrene in the peroxidative reaction. Nervous membranes are particularly sensitive to lipid oxidation which differentially acts on the two layers of the membrane determining a greater rigidity of the exofacial one. Thus, we consider the possibility to asymmetrically introduce the pyrene ring, as P12-FA or P12-CS, in synaptosomes for monitoring lipid peroxidation in each layer of the membrane. The amount of the two probes incorporated in the membrane was 20 +/- 3 and 10 +/- 2 nmol/mg of protein for P12-FA and P12-CS, respectively. P12-FA was symmetrically distributed in the two layers, whereas 95% of P12-CS was incorporated in the exofacial layer of the membrane as determined by TNBS measurements. The decrease in fluorescence of synaptosome associated pyrene was, in the early stages of lipid peroxidation, greater for P12-CS than for P12-FA labelled membranes, indicating a greater susceptibility of the exofacial layer to iron-induced peroxidation.

Animals

Age-related differences in synaptosomal peroxidative damage and membrane properties.

Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.

Aging

Studies on peroxidation processes of model membranes and synaptosomes: role of phosphatidic acid.

Dipalmitoylphosphatidic acid (DPPA) was found to exert a strong inhibitory effect on Fe-induced peroxidation of arachidonic acid inserted into liposomal dipalmitoylphosphatidylcholine (DPPC) vesicles. This inhibition was quite effective both below and above the phase transition temperature of the liposomes. Moreover, we demonstrated the antiperoxidative activity of phosphatidic acid (PA) in synaptosomal membranes. PA enriched synaptosomes were prepared by the stimulation of the endogenous phospholipase D activity or by the incubation of the synaptosomes with Streptomyces chromofuscus phospholipase D. The possible contribution of PA to the in vivo defense mechanism against free radical-induced damage is discussed.

Animals

Studies on peroxidation processes of model membranes: role of pyrophosphate.

Phosphate-containing compounds of both a lipophilic (dipalmitoylphosphatidic acid, lysophosphatidic acid and dicetylphosphate) and hydrophilic nature (orthophosphate, 3-phosphoglyceric acid, 2,3-diphosphoglyceric acid and pyrophosphate in particular) have been found to inhibit to varying degrees the lipoperoxidation of liposomal arachidonic acid. Lipophilic compounds seem to act exclusively at the lipid component level of the membrane, giving rise to polyanionic complexes with free arachidonic acid or its radical derivatives that could bind the Fe2+ strongly (thereby inhibiting the iron pro-oxidation activity) or to minimize the lateral mobility of the lipid radicals (thereby reducing the propagation of lipid peroxidation). The high antiperoxidative power of hydrophilic compounds, and in particular of pyrophosphate, must, on the contrary, be primarily attributed to their ability to form very stable complexes with the Fe present in the solution surrounding the liposomal membranes. The possible contribution of these physiological compounds to the in vivo defense mechanism against radical-induced damage is discussed.

Arachidonic Acid

Studies on peroxidation of arachidonic acid in different liposomes below and above phase transition temperature.

The mechanism of Fe-induced peroxidation of arachidonic acid (AA) in small unilamellar vesicles (SUV) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) was studied below and above gel to liquid-crystalline phase transition temperature (Tm). In both liposomes the AA peroxidation resulted higher in the temperature range below Tm, but the extent of malonyldialdehyde (MDA) formation was dramatically lower in DPPE vesicles when compared with corresponding DPPC liposomes. A possible explanation for this is discussed.

Arachidonic Acids

N-pyrene dodecanoyl sulfatide as membrane probe: a study of glycolipid dynamic behavior in model membranes.

An N-linked pyrene-dodecanoyl sulfatide was employed to measure the ratio of excimer fluorescence to monomer fluorescence intensities (E/M). The E/M values provided information about both the dynamic behavior and the structural distribution of the labelled glycolipid in note dispersion of micellar sulfatides and multilamellar vesicles of different phospholipids. Most of the labelled sulfatide seems to be located in domains sequestered from the surrounding phospholipids still above the phase transition temperature of the vesicles. The glycolipids sequestered in these domain environments are less sensitive to the structural changes that the addition of cholesterol or Ca2+ can induce in the phospholipid regions during the phase transition.

Fluorescent Dyes

Erythrocyte D-glucose transport activity in reconstituted model membranes of different lipid composition.

The stereospecific influx of D-glucose into liposomes formed on sonication of different glyco- and phospholipids with transport proteins from human erythrocyte ghosts solubilized with Triton x-100 was measured as an index of their total D-glucose transport activity. Specific D-glucose transport increased when acidic phospho- and glycolipids (especially sulfatide) were added to the phosphatidylcholine bilayers of the model membranes while cholesterol strongly inhibited the process. The modulation of D-glucose transport activity and its possible correlation with the lipid composition and the chemico-physical state of the erythrocytes is discussed.

Cerebrosides

Interactions of insulin with sulfatide-containing vesicles of phosphatidylcholine at different pHs.

Positively charged insulin is described to induce aggregation of phosphatidylcholine vesicles containing 10 mol% sulfatide at acidic pH. Techniques including light-scattering, Sepharose chromatography, centrifugation, trapped volume determination, circular dichroism and fluorescence polarization, demonstrate that large amounts of negatively charged insulin remain firmly associated to the vesicles upon raising the pH to 7. This is surprising, since only trace amounts of insulin associate to the sulfatide-containing vesicles upon direct incubation at pH 7. The possible molecular explanation of the phenomenon and the relevance of these findings to the actions of insulin in vivo are discussed.

Circular Dichroism

Preparation of spin-labelled sulfatides for EPR studies on model membranes.

Using the N-hydroxysuccinimide ester of the fatty acids, galactosylceramide I3-sulfate containing a 5-or 16-doxyl-stearoyl residue was prepared in good yield by acylation of the galactosylsphingosine I3-sulfate (lysosulfatide) obtained from the saponification of the bovine brain sulfatide. The EPR behavior of the two semisynthetic sulfatides was analyzed in natural sulfatide micelles and in multilamellar vesicles of egg phosphatidylcholine. The evaluated parameters demonstrate that these spin-labelled sulfatides can be used for the study of sulfatide behavior in lipid structures.

Animals

Fluorospectroscopic studies of mixtures of distearoylphosphatidylcholine and sulfatides with defined fatty acid compositions.

Simple study models characteristic for lamellar organization of distearoylphosphatidylcholine and sulfatide have been prepared for fluorospectroscopic investigations on the influence of these glycolipids on the chemico-physical properties of lecithin bilayers. The motion of 1,6-diphenyl-1,3,5-hexatriene in mixed lecithin-sulfatide bilayers changed with temperature, with the compositional ratio of the two lipids, with the presence of divalent cations such as Ca2+ and with the fatty acid composition of sulfatide moiety. Steady-state fluorescence measurements of the average motion of the fluorophore permit evaluation of the gel to liquid-crystalline phase transition in all these membrane models containing different sulfatides.

Fatty Acids

Calorimetric properties of mixtures of distearoylphosphatidylcholine and sulfatides with definite fatty acid composition.

The thermotropic behavior of multilamellar liposomes prepared from mixtures of distearoyllecithin and sulfatide of definite fatty acid composition, has been studied by DTA calorimetry. The saturated stearoyl- and lignoceroyl-sulfatides do not modify significatively both the delta H and the Tm of the phase transition of the lecithins. These parameters, on the contrary, are decreased by all the sulfatides containing unsaturated fatty acids. A contrasting effect is at last exhibited by alpha-OH-stearoylsulfatide which promotes a decrease in delta H but not in Tm of the phase transition. The possibility that these sulfatide properties can influence the dynamic interactions and the sovramolecular organization of the phospholipid bilayers has been discussed.

Calorimetry

Synthesis of N-acetylcysteine compounds.

The preparation of some anilides, esters and amides of N-acetyl and N,S-diacetyl-cysteine is described. Of the compounds synthesized, particular interest was aroused by the p-hydroxyanilide derivatives of N-acetyl and N,S-diacetyl-L-cysteine owing to their analgesic, antiinflammatory and antipyretic activity.

Acetylcysteine

In vitro and in vivo studies with anionic sulfatide-liposomes containing adriamycin.

Neutral and negatively charged liposomes containing Adriamycin (ADM) were examined for efficiency of drug entrapment and stability in serum. The greatest entrapment of ADM was obtained with negatively charged liposomes containing sulfatide. Moreover, these sulfatide-containing liposomes were more stable than other liposomes in the presence of serum. Tissue distribution studies indicated that the levels of ADM were increased several-fold in mouse liver and spleen after i.v. injection of the drug entrapped in sulfatide-liposomes, while levels of the drug were significantly diminished in the heart. Finally, the in vivo antitumor activity of ADM in liposomes containing sulfatides resulted in significantly greater survival rates than free ADM or ADM entrapped in liposomes without sulfatides.

Animals