Biomedical subjects

M Masserini

Publications and source records attributed to M Masserini.

At least 37 records · Page 2Linked to original sources

PubMed1995

Spontaneous transfer of GM3 ganglioside between vesicles.

The spontaneous transfer between membranes of GM3, a ganglioside present in a vesicular form of aggregation instead of micellar form like the majority of gangliosides in aqueous medium, has been studied. Upon incubation of GM3 in the presence of dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles at 50 degrees C, mixed GM3/DPPC vesicles are formed. The maximum amount of GM3 that can be inserted into vesicles is about 8%. The temperature dependence of the kinetics has been followed by the excimer formation technique, using the fluorescent analogue pyrenyldodecanoyl-GM3. The transfer of ganglioside from its vesicles to DPPC vesicles depends on the physicochemical characteristics of both the donor and of the acceptor vesicles and increases with the temperature (k = 0.006 0.012, 0.037 at 30, 41 and 50 degrees C, respectively), with a major break point at 41 degrees C and a minor one at 35 degrees C. These temperatures correspond to the gel- to liquid-crystalline transition of DPPC (Tm = 41.3 degrees C), and to a temperature transition displayed by GM3 ganglioside. Similar experiments performed with erythrocyte ghosts yielded a rate constant of 0.04 at 37 degrees C. For the transfer of ganglioside from DPPC (donor) to DMPC (acceptor) the rate constants were 0 at 15 degrees C (both phospholipids in the gel phase), 0.005 at 37 degrees C (donor in the gel phase, acceptor in the fluid phase) and 0.04 at 50 degrees C (both phospholipids in the fluid phase).(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Effects of chronic ethanol exposure on cultured cerebellar granule cells.

The aim of this study was to investigate the lipid content and composition of rat cerebellar granule cells grown in the presence of ethanol (40, 55, or 80 mM) during in vitro differentiation. Quantitative analyses showed no effects of 40 mM ethanol, whereas a significant increase of total cholesterol was observed at 55 mM. Cells exposed to the highest ethanol dose (80 mM) were characterized by a higher sialidase activity, and by the modification of the ganglioside pattern and phospholipid fatty acid composition. The observed modifications were accompanied by changes of membrane anisotropy fluorescence assessed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene.

Animals↗

PubMed1994

Exposure to galactose oxidase of GM1 ganglioside molecular species embedded into phospholipid vesicles.

The exposure of GM1 molecular species present in the native ganglioside, carrying C18:1 or C20:1 long-chain bases (LCB), to Dactylium dendroides galactose oxidase was studied. When native GM1 (49.3% C18:1 and 50.7% C20:1 LCB, respectively), was inserted in dipalmitoylphosphatidylcholine vesicles and partially oxidized (10%), the proportion of C18:1 and C20:1 species in the oxidized GM1 was 59.6% and 40.4%, respectively, suggesting a preferential action of the enzyme on the shorter species. The Vmax of the enzyme was higher on C18:1 GM1 than on C20:1 GM1. The molecular species were affected without any preference after partial (10%) oxidation of GM1 incorporated in egg phosphatidylcholine vesicles or in micellar form. These data indicate that the exposure of the terminal galactose moiety of GM1 ganglioside to galactose oxidase is affected by the ganglioside ceramide composition as well as the phospholipid environment, that presumably determine the distribution (molecular dispersion, segregation) of the ganglioside within the membrane.

Animals↗

PubMed1994

Modifications of low density lipoprotein induced by the interaction with human plasma glycosaminoglycan-protein complexes.

Glycosaminoglycan (GAG)-protein complexes from human plasma were separated into low charge (LC-GP) and high charge (HC-GP) components. LC-GP and HC-GP differed with respect to GAG and protein composition and to molecular size. The in vitro interaction of both GAG-protein complexes with human LDL was investigated. LC-GP did not precipitate LDL. On the contrary, HC-GP formed insoluble complexes with LDL, following a biphasic behaviour on increasing HC-GP concentration. In the presence of a HC-GP/LDL ratio higher than 0.02 the interaction stoichiometry was shifted towards the formation of soluble complexes. Papain treatment of HC-GP completely prevented LDL precipitation. Moreover, the extent of HC-GP-induced precipitation of LDL was markedly reduced by the simultaneous addition of LC-GP. Data obtained with standard GAGs showed that heparin (HE) and chondroitin-6-sulphate (C6S) were the most effective ligands in precipitating LDL. However, the shape of precipitation curves was markedly different. C6S behaved similarly to HC-GP, suggesting that GAG chains could play an important role in insoluble complex formation with LDL. Steady-state fluorescence anisotropy investigation indicated that HC-GP induced a significant decrease in the microviscosity of LDL hydrophobic region. This effect was no longer detectable after either addition of LC-GP or papain treatment of HC-GP. Differential scanning calorimetry (DSC) demonstrated that both lipid and protein components of LDL were affected by the interaction with HC-GP. The temperature of irreversible thermal unfolding of apo B100 was shifted to a lower value and a second peak appeared in the region of the reversible melting of cholesterol esters. Both the fluorescence anisotropy and the DSC data obtained with standard HE and C6S indicated that GAG chains were directly involved in affecting physico-chemical properties of complexed LDL. These results suggest that the interaction with plasma HC-GP could modify LDL structural properties. However, LC-GP is likely to act as a modulator, probably preventing the interaction between HC-GP and circulating LDL.

Blood Proteins↗

PubMed1994

Protective effect of some exogenous glycolipids on human cultured keratinocytes against lipid peroxidation.

Cultured human keratinocytes were incubated in the presence of glycolipids (GM1, asialoGM1, glucosylceramide and galactosylceramide) added to the medium at a concentration of 10(-5) M. All of them became associated with the cells, but in different amounts depending on the type of glycolipid and on the time in culture. GM1- and asialoGM1-treated keratinocytes developed a resistance to peroxidative stress induced by FeSO4/H2O2 treatment. These results suggest that these exogenously added glycolipids could play a role as eutrophic, repairing and protective agents for the epidermis.

Cells, Cultured↗

PubMed1994

Synthesis and aggregative properties of GM1 ganglioside (IV3Neu5AcGgOse4Cer) containing D-(+)-2-hydroxystearic acid.

GM1 ganglioside containing a hydroxylated fatty acid moiety, GM1(OH), was synthesized starting from lyso-GM1 and D-(+)-2-hydroxystearic acid. The aggregative, geometrical and distribution properties of GM1(OH) were compared with those of stearic acid containing GM1 ganglioside; laser light scattering measurements, differential scanning calorimetry and fluorescence spectroscopy were used. GM1 and GM1(OH) are present in solution as micelles with a hydrodynamic radius of 58.7 and 60.0 A, and molecular mass of 470 and 570 kDa, respectively. The surface area occupied by the monomer of GM1(OH) at the lipid-water interface of the aggregate was calculated to be 117 A2, which is 3 A2 lower than that determined for GM1. Proton NMR analyses of GM1 and GM1(OH) suggest different three-dimensional structures at the ganglioside lipid-water interface. Both GM1(OH) and GM1 inserted into dipalmitoylphosphatidylcholine (DPPC) vesicles undergo segregation phenomena, with the formation of ganglioside-enriched microdomains, but GM1(OH) shows a higher degree of dispersion in the DPPC matrix and exerts a lower rigidifying effect than does GM1.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Fluorescence excimer formation imaging: a new technique to investigate association to cells and membrane behavior of glycolipids.

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.

Animals↗

PubMed1993

Characterisation of biotinylated liposomes for in vivo targeting applications.

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550-DPPE or PEG2000-DPPE in liposomes containing biotin-DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin-DPPE liposomes containing GM1. Biodistribution studies in C26 tumour-bearing mice showed that GM1-liposomes containing small amounts of biotin-DPPE have long circulation life-times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin-DPPE were detected within extra-vascular spaces in tumours. In addition it was shown that biotin-DPPE in GM1-liposomes bound streptavidin in situ. These results suggest that GM1-liposomes containing biotin-DPPE have potential use as diagnostic or therapeutic reagents in pre-targeting applications dependent on the high-affinity interaction of biotin with streptavidin.

Animals↗

PubMed1993

Interaction of glycosphingolipids and glycoproteins: thermotropic properties of model membranes containing GM1 ganglioside and glycophorin.

High-sensitivity differential scanning calorimetry (DSC) was used to study the mutual interactions between a glycoprotein (human glycophorin, GPA) and a glycosphingolipid (GM1 ganglioside) embedded in large unilamellar vesicles composed of dimyristoylphosphatidylcholine (DMPC). The DSC thermograms exhibited by DMPC/GM1 vesicles, either in the presence or in the absence of GPA, are resolvable into two components. The relative contribution of the minor component, centered at higher temperature, to the total enthalpy and its transition temperature increase with the concentration of the glycolipid embedded in the vesicles. This minor peak, undetectable in the absence of ganglioside, is indicative of the occurrence of lateral phase separation and suggests that GM1 ganglioside-enriched domains are present within the bilayer. At a given concentration of GM1 embedded in the vesicles, the proportion of the phase-separated peak is higher in the presence of GPA, suggesting that the glycoprotein enhances the tendency of GM1 to segregate. Experiments investigating the thermotropic behavior of GPA show that the temperature of irreversible thermal unfolding of the glycoprotein inserted in DMPC vesicles, centered at 65.9 degrees C in the absence of GM1, is shifted to 57.6 degrees C when GM1 is present in the bilayer. These results indicate that, at least in this experimental system, on the one hand, GPA enhances the tendency of the glycolipid to segregate within the membrane, and on the other hand, the glycolipid clusters affect the protein conformation and oligomerization in the membrane.

Calorimetry, Differential Scanning↗

PubMed1993

Action of alpha-L-fucoside from Octopus vulgaris hepatopancreas on phospholipid vesicles containing the fucosylated ganglioside FucGM1.

The behaviour of a highly purified alpha-L-fucosidase (E.C. 3.2.1.51) extracted from octopus hepatopancreas was studied with phospholipid vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS) containing the fucosylated ganglioside FucGM1, a potential natural substrate of the enzyme. The substrate recognition and hydrolysis take place only with PS/FucGM1 mixtures via an association process of the enzyme with the vesicles at acidic pH; the enzyme rapidly and stably binds to PS vesicles but not to PC vesicles. The data suggest that only the PS-associated enzyme is able to hydrolyse FucGM1 embedded in the same bilayer. The enzyme association with FucGM1/PS vesicles is a prerequisite for ganglioside hydrolysis but is followed by irreversible enzyme inactivation.

Animals↗

PubMed1993

Age-related changes in the ceramide composition of the major gangliosides present in rat brain subcellular fractions enriched in plasma membranes of neuronal and myelin origin.

Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1a, GD1b, GT1b, and GQ1b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20:1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80-90% vs. 60-70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.

Aging↗

PubMed1992

Lipid interaction of tetanus neurotoxin. A calorimetric and fluorescence spectroscopy study.

The interaction of Tetanus toxin with phospholipid vesicles containing gangliosides (GD1a, GD1b or GT1b) or phosphatidic acid has been investigated at neutral or acidic pH. Change in the thermotropic properties of the vesicles occurred only after addition of the toxin at acidic pH, and led to surface binding or membrane insertion of the protein, dependent on the physical state of the membrane. Most remarkably, toxin addition at acidic pH to dipalmitoyl-phosphatidylcholine vesicles containing GT1b ganglioside, caused formation of ganglioside microdomains on the vesicle surface.

Calorimetry, Differential Scanning↗

PubMed1992

Fuc-GM1 ganglioside mimics the receptor function of GM1 for cholera toxin.

The ability of Fuc-GM1 ganglioside to mimic the receptor function of GM1 for cholera toxin (CT) has been investigated. For this purpose, rat glioma C6 cultured cells were enriched with Fuc-GM1 and the responsiveness to CT was compared with that of cells enriched with GM1 ganglioside. Fuc-GM1 was taken up by cells as rapidly and to the same extent as GM1. When comparable amounts of ganglioside were associated, the cells enriched with Fuc-GM1 bound the same amount of 125I-CT as did cells enriched with GM1. Under conditions in which GM1- and Fuc-GM1-enriched cells bound comparable amounts of CT, the Fuc-GM1-treated cells accumulated virtually the same amount of cyclic AMP as did GM1-treated cells, and activation of adenylate cyclase was also similar. The lag time preceding the CT-induced cAMP accumulation was the same in Fuc-GM1- and GM1-enriched cells. High-sensitivity isothermal titration calorimetry (ITC) experiments showed that the association constants of CT with Fuc-GM1 or GM1 ganglioside were comparable (4 x 10(7) M-1 and 1.9 x 10(7) M-1, respectively, at 25 degrees C). Also, the association constants of the B-subunit pentamer with Fuc-GM1 or GM1 ganglioside were comparable (about 3 x 10(7) M-1 and 7 x 10(7) M-1, respectively, at 25 degrees C).

Adenylyl Cyclases↗

PubMed1992

Thermotropic behavior of fatty acid ethyl esters in phospholipid liposomes.

The thermotropic behavior of a series of synthetic fatty acyl ethylesters (FAEE) in multilamellar liposomes has been studied by differential scanning calorimetry and monitoring the changes in polarization emitted by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Their thermotropic behaviour has been compared to that of the homologous fatty acids (FA) from which they are synthesized in vivo in the presence of ethanol. Compared to the correspondent FA, saturated FAEE show, depending on the chain length, a minor rigidifying effect or even a slight fluidizing effect on phospholipid bilayers. Unsaturated FAEE show, compared to the homologous FA, slightly greater fluidizing properties. The difference between FA and FAEE is more evident in single component phospholipid liposomes in the gel phase, and in mixed liposomes of two lipids at temperatures at which microdomains of gel and liquid zones coexist. The calorimetric data suggest that FAEE are completely miscible with phospholipids both in the gel and liquid phases; they appear to destabilize the bilayer wherein the ethoxy head group interferes with the intrinsic phospholipid interactions.

Animals↗

PubMed1991

Role of phosphatidylethanol in membranes. Effects on membrane fluidity, tolerance to ethanol, and activity of membrane-bound enzymes.

We investigated the effect of phosphatidylethanol (PEt) on fluidity and membrane tolerance to the fluidization induced by ethanol as well as on the activity of two membrane-bound enzymes, Na+/K+ ATPase and 5'-nucleotidase. PEt was synthesized from 1,2-dimyristoylphosphatidylcholine and phosphatidylcholine from bovine brain and studies were performed to determine the optimal experimental conditions for the insertion of PEt in natural bilayers. The effects of PEt, evaluated by differential scanning calorimetry or fluorescence polarization techniques, were studied in model membranes made of synthetic phospholipids or made of total lipids extracted from rat brain crude mitochondrial fraction (P2 fraction) and from natural membranes (P2 fraction). The presence of PEt increased the fluidity of artificial as well of natural membranes, but tolerance to the addition of ethanol, displayed by dimyristoylphosphatidylcholine vesicles and by natural membranes containing PEt, was lacking in vesicles made of dimyristoylphosphatidylethanolamine and in artificial bilayers reconstituted from total P2 lipid extracts, suggesting an involvement of PC on PEt-induced ethanol resistance. Na+/K+ ATPase activity was enhanced by the addition of small amounts of ethanol (up to 50 mM) and progressively inhibited at higher concentrations, while 5'-nucleotidase was not affected up to 400 mM ethanol. The presence of PEt in the bilayer exerted the opposite effects on the two enzymes, reducing the Na+/K+ ATPase activation induced by ethanol and enhancing 5'-nucleotidase activity. The mechanisms of the PEt-induced modifications are discussed.

5'-Nucleotidase↗

PubMed1991

Evidence for nonrandom distribution of GD1a ganglioside in rabbit brain microsomal membranes.

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long-chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a (approximately 9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long-chain-base analysis showed that newly-formed GM1 contained approximately 68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing approximately 10% hydrolysis. Long-chain-base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipalmitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long-chain base are more molecularly dispersed than those containing C18:1 long-chain base.

Animals↗

PubMed1990

Cyclic AMP accumulation in HeLa cells induced by cholera toxin. Involvement of the ceramide moiety of GM1 ganglioside.

The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1. The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate. After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored. Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species. Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1. Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.

Animals↗

PubMed1990

Effect of gangliosides on membrane permeability studied by enzymic and fluorescence-spectroscopy techniques.

The effect of gangliosides on membrane permeability was investigated by studying the kinetic properties of cytochrome c oxidase, the activity of which, when the enzyme is reconstituted in phospholipid vesicles, is dependent on membrane permeability to H+ and K+. The experiments indicate that three different gangliosides (GM1, DD1a, GT1b) incorporated into cytochrome c oxidase-containing phospholipid vesicles stimulate enzymic activity, in the absence of ionophores, most probably by disorganizing the bilayer lipid assembly and increasing its permeability to ions. This interpretation was confirmed by fluorescence-spectroscopy experiments in which the rate of passive leakage of carboxyfluorescein entrapped in the vesicles was measured. Cholera toxin, or its isolated B-subunit, added to GM1-containing proteoliposomes inhibited cytochrome c oxidase activity, indicating the lack of formation, under these experimental conditions, of channels freely permeable to H+ or K+.

Electron Transport Complex IV↗