Biomedical subjects

M Masserini

Publications and source records attributed to M Masserini.

At least 19 recordsLinked to original sources

PubMed2007

Changes in the composition of detergent-resistant membrane domains of cultured neurons following protein kinase C activation.

Changes in the composition of cell fractions, and in particular of detergent-resistant membranes (DRM) isolated from cultured rat cerebellar granule cells, were taken as possible changes in lipid raft composition during a signal transduction event. After activation of protein kinase C (PKC) with phorbol esters (PMA) or glutamate, the content of PKC and of proteins highly enriched (GAP43, Fyn, and PrP(c)) or not (MARCKS) in DRM was followed. PKC activation strongly increased its association with membranes (from 2% to 75%), causing its enrichment within DRM; the substrate GAP43, enriched in DRM, remained membrane associated, but its proportion in DRM dramatically decreased (from about 40% to 2.5%), suggesting its shift from raft to nonraft membranes, possibly as a consequence of phosphorylation by PKC. The distribution of Fyn and PrP(c) (DRM-enriched) and of MARCKS (present mainly outside DRM) did not change. PKC activation was followed by an increase of GAP43 and MARCKS phosphorylation (about 7- and 8-fold, respectively). Noteworthy was that, after cell treatment with the lipid raft-disrupting drug methyl-beta-cyclodextrin, PKC activation occurred normally, followed by MARCKS phosphorylation, but GAP43 phosphorylation did not occur. Taken altogether, these data suggest that the integrity of lipid rafts is necessary for PKC to affect GAP43 and catalyze its phosphorylation.

Animals↗

PubMed2001

Role of sphingolipids in the biogenesis of membrane domains.

In recent years, a huge interest in sphingolipid- and cholesterol-enriched membrane domains has risen, after their involvement in fundamental membrane-associated events such as signal transmission, cell adhesion and lipid/protein sorting was postulated. Theoretical considerations and several experimental data suggest that sphingolipids play an important role in the biogenesis and function of domains. In fact, their physicochemical features, different from those of other membrane lipids, allow their interaction either with other sphingolipids or with other membrane components and external ligands. Owing to these features, sphingolipids may undergo segregation and represent a nucleation point for co-clustering with other lipids and proteins in a complex, functional domain. Moreover, sphingolipids confer dynamic properties on domains, a fundamental feature for the modulation of their postulated functions.

Animals↗

PubMed2001

Thermal stability of the hemagglutinin-neuraminidase from Sendai virus evidences two folding domains.

The domain structure of hemagglutinin-neuraminidase from Sendai virus (cHN) was investigated by studying the thermal stability in the 20-100 degrees C range. Differential scanning calorimetry evidences two conformational transitions. The first transition is apparently a reversible two-state process, with Tm 48.3 degrees C, and is shifted to 50.1 degrees C in the presence of the substrate analogue 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid, meaning that the substrate binding domain is involved in the transition. The second transition, with apparent Tm 53.2 degrees C, is accompanied by irreversible loss of enzymatic activity of the protein, and the presence of the substrate analogue does not affect the Tm. The data indicate that cHN is composed of two independent folding domains, and that only one domain is involved in the binding of the substrate. Our results suggest that the paramyxovirus neuraminidases have the folding properties of a two-domain protein.

Calorimetry↗

PubMed2000

Tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane.

After incubation of intact living cultured rat cerebellar granule cells at 37 degrees C with a new GM1 ganglioside analog, carrying a diazirine group and labeled with (125)I in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins were detected in a membrane-enriched fraction. A protein of about 55 kDa with a pI of about 5 carried a large portion of the radioactivity even if incubation and cross-linking were performed at 4 degrees C and in the presence of inhibitors of endocytosis, suggesting that it is cross-linked at the plasma membrane. Immunoprecipitation and Western blotting experiments showed the positivity of this protein for tubulin. Trypsin treatment of intact cells ruled out the involvement of a plasma membrane surface tubulin. Release of radioactivity from cross-linked tubulin after KOH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked fatty acid anchor of tubulin. Low buoyancy, detergent-resistant membrane fractions, isolated from cells after incubation with the GM1 analogue and photoactivation, proved their enrichment in endogenous and radioactive GM1 ganglioside, sphingomyelin, cholesterol, signal transduction proteins, and tubulin. It is noteworthy that radioactive tubulin was also detected in this fraction, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma membrane. Parallel experiments carried out with a phosphatidylcholine analogue, also carrying a diazirine group and labeled with (125)I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. Taken together, these results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane domains can provide a key for a better understanding of their biological role.

Animals↗

PubMed2000

Effect of ethanol amine plasmalogens on Fe-induced peroxidation of arachidonic acid in dipalmitoylphosphatidylcholine vesicles.

We have investigated the influence of ethanolamine plasmalogens on iron-induced oxidation of arachidonic acid in dipalmitoylphosphatidylcholine (DPPC) vesicles. Lipoperoxidation was induced by the addition of 50 microM FeSO4 and studied above (50 degrees C) and below (15 degrees C) the gel-to liquid transition temperature of the vesicles, at two different pH values (7.4 or 6.4). The extent of peroxidation was measured as thiobarbituric reactive product formed and the influence exerted by ethanolamine plasmalogens (PEPL) in this process was compared to that of dipalmitoylphosphatidylethanolamine (DPPE) and diacylphosphatidylethanolamines (DAPE). The extent of peroxidation of arachidonic acid embedded in DPPC vesicles was similar at the two temperatures and greater at 50 degrees C under acidic conditions. However, the peroxidative process was significantly decreased at 50 degrees C in the presence of PEPL, but not of DPPE or DAPE and the inhibitory effect was enhanced at pH 6.4. The possibility that a different phase distribution of the phospholipids, namely a transition from a lamellar to a hexagonal phase, may play a role in the scavenger effect of ethanolamine plasmalogens is discussed.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

Dynamics of glycolipid domains in the plasma membrane of living cultured neurons, following protein kinase C activation: a study performed by excimer-formation imaging.

Dynamic changes of glycolipid domains within the plasma membranes of cultured rat cerebellar granule cells have been investigated. For this purpose, a pyrene-labelled derivative of G(M1) ganglioside has been incorporated in the cell plasma membrane, and the rate of excimer formation, directly related to the formation of domains, has been studied by a fluorescence imaging technique (excimer-formation imaging). Fluorescence imaging showed that upon addition of 100 microM glutamate, indirectly inducing the activation of protein kinase C (PKC), glycolipid concentration within domains increases in cell bodies. Comparable effects were exerted by the addition of PMA, directly inducing the activation of PKC. On the contrary, the phorbol ester was not effective in the presence of the specific PKC inhibitor, bisindolylmaleimide. These results suggest that glycolipid-enriched domains are dynamic supramolecular structures affected by membrane-associated events, such as PKC activation. Dynamic changes of domains could be important in modulating their postulated participation in a series of functions, including signal transduction and lipid/protein sorting.

Animals↗

PubMed1999

Interaction of liposomes composed of phospholipids, GM1 ganglioside and cholesterol with human keratinocytes in culture.

We studied the possibility of supplementing human keratinocytes with exogenous lipids (phospholipids, sphingolipids and cholesterol) and evaluated their influence on cell proliferation, using cells cultured in vitro. Experiments carried out with liposomes composed of cholesterol/GM1 ganglioside and different phospholipids (5:1.5:10, M/M/M), showed that liposomes associated with cells more efficiently when they contained soya lecithin. The treatment with liposomes made of the ternary mixture did not modify the rate of cell proliferation, as assessed by the incorporation of [3H]-thymidine. In contrast, the proliferation rate strongly decreased (65% with respect to the control) using the same liposomes without GM1. Experiments carried out with GM1 alone showed a strong stimulation of the proliferation rate (144% with respect to the control). Fluorescence dequenching experiments, carried out with the probe octadecyl rhodamine B chloride, showed that fusion was the main mechanism of liposome-cell interaction. Metabolic studies established that exogenously administered GM1--either embedded in liposomes or as a pure glycolipid dispersion--led to the production of several products, including ceramide. Altogether, these results show that different, opposing effects can be exerted on cell proliferation by the administration of lipids, separately or in mixtures, to human keratinocytes, and indicate the importance of a correct formulation for supplementing human keratinocytes with exogenous lipids.

Cells, Cultured↗

PubMed1999

Involvement of glycolipid-enriched domains in the transduction mechanism of neurotrophins in cultured neurons.

Specialized domains, displaying a peculiar lipid and protein composition, are present within the plasma membrane of mammalian cells and play a pivotal role in fundamental membrane-associated events. Among lipids, sphingolipids (in particular glycolipids and sphingomyelin) are characteristically enriched within such domains. Moreover, a series of functionally related proteins is present, suggesting the involvement of these membrane structures in the mechanism of signal transduction and lipid/protein sorting. An increasing body of evidence suggests that domains are dynamic structures, and that their dynamic fluctuations can modulate the activity of domain-associated proteins through changes of glycolipid protein interaction. Even if a large body of experimental investigation has been carried out on eukaryotic cells, only little attention has been paid to the neuron. The purpose of the present review is to summarize the observations implying a functional role of glycolipid-enriched domains in cultured rat cerebellar granule cells.

Animals↗

PubMed1999

Glycolipid-enriched caveolae and caveolae-like domains in the nervous system.

Recent years have been characterized by a booming interest in research on caveolae and caveolae-like membrane domains. The interest in this subject grew further, when their involvement in fundamental membrane-associated events, such as signal transmission and lipid/protein sorting, was postulated. Substantial progress has been reached in understanding the biological role of membrane domains in eukaryotic cells. The neuron, however, which perhaps represents one of the greatest challenges to research on membrane traffic and function, has only been partially investigated. The purpose of the present review is to survey this issue in the nervous system. We confine ourselves to the presence of membrane domains in the nervous system and discuss this in the context of three facts: first, glycolipids are peculiarly enriched in both caveolae and caveolae-like domains and are particularly abundant in the nervous system; second, the neuron is characterized by a basic dual polarity, similar in this respect to other polarized cells, where the role of glycolipid-enriched domains for lipid/protein sorting has been better ascertained; and third, neurons evolved from, and are related to, simpler eukaryotic cells, allowing us to find analogies with more investigated nonneuronal cells.

Cell Membrane↗

PubMed1999

Exogenous administration of gangliosides displaces GPI-anchored proteins from lipid microdomains in living cells.

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid-cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4 degrees C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid-cholesterol microdomains in living cells.

Animals↗

PubMed1998

Influence of endogenous GM1 ganglioside on TrkB activity, in cultured neurons.

We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain-derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol-12-myristate-13-acetate (1 microM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain-derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.

Animals↗

PubMed1998

Change of ganglioside accessibility at the plasma membrane surface of cultured neurons, following protein kinase C activation.

While the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degreesC), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 microM glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 microM glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state.

Animals↗

PubMed1998

Glycolipid-protein interaction in the mechanism of signal transduction: studies with a photoactivable ganglioside analogue.

An increasing body of evidence suggests that glycolipid domains are present on the plasma membrane surface of mammalian cells and play a key role in signal transduction. We have investigated the modulation of glycolipid-protein interaction consequent to a specific event occurring at the plasma membrane. For this purpose, a new photoactivable, radioactive derivative of GM1 ganglioside, carrying a phenyldiazirine fatty acid labelled with 125I, has been used with rat cerebellar granule cells in culture. Upon incubation of photoactivable GM1 with the cells followed by illumination, several proteins become radioactive and were detectable on the two dimensional-electrophoresis, which points to their interaction with the ganglioside. Upon addition of cytotoxic doses of glutamate, known to induce indirectly the activation of protein kinase C (PKC), one of the proteins crosslinked by photoactivable GM1 in control cells of molecular mass about 92 kDa and pI about 4, was not anymore detectable; this suggests its exclusion from the glycolipid domains. On the contrary, another protein, of about 15 kDa and pI 6.5, previously not crosslinked, was interacting with the ganglioside derivative after glutamate treatment. Comparable effects were exerted by phorbol-2-myristate-3-acetate, which directly induces the activation of PKC. These results show that PKC activation, a key step of inbound trans-membrane signalling, affects the interaction between glycolipids and proteins at the plasma membrane surface, possibly within a mixed domain. The dynamic modulation of ganglioside-protein interaction may affect the involvement of glycolipid domains in membrane-located events such as signal transmission and lipid/protein sorting.

Animals↗

PubMed1997

Ganglioside lateralization in the brain of female rats.

The ganglioside composition of the cerebral hemispheres of young and adult rats of either sex has been herein assessed for the first time. In females, the total ganglioside content at any age, the content of GM1, GD1a, and GD1b at 8 days, and the content of GM1, GD1b, GT1b, and GQ1b at 60 days were higher in the right than in the left hemisphere. In males, no difference was observed. Concerning the ceramide moiety, a difference was displayed by C18:1 long-chain base in GD1a, whose proportion was higher in the left than in the right hemisphere of females aged 8 days. The comparison between homolateral hemispheres of rats of different sex revealed several differences. On average, in 8-day-old animals, the content of gangliosides was higher in females than in males. At 60 days the amount of gangliosides was on average lower in females than in males, even if with some exception. The data obtained with the current investigation show the existence of a ganglioside lateralization in rat brain, exclusively in females, and almost entirely at charge of the oligosaccharide portion. Moreover, age-dependent changes of ganglioside pattern and content show a dependence on brain lateralization.

Aging↗

PubMed1997

Lipid domains in the membrane: thermotropic properties of sphingomyelin vesicles containing GM1 ganglioside and cholesterol.

The thermotropic behavior of palmitoylsphingomyelin vesicles containing GM1 ganglioside and cholesterol has been investigated by high-sensitivity differential scanning calorimetry. The thermograms exhibited by binary palmitoylsphingomyelin/GM1 mixtures are resolvable into two components. The relative contribution of the minor component, undetectable in the absence of ganglioside, to the total enthalpy and its transition temperature (>40 degrees C) increase with the concentration of the glycolipid embedded in the vesicles. These data suggest the occurrence of lateral phase separation and that more ordered, higher melting GM1 ganglioside-enriched domains are present within the sphingomyelin bilayer. Studies on binary sphingomyelin/cholesterol mixtures confirmed the known tendency of the sterol to decrease the total enthalpy of sphingomyelin, forming cholesterol-enriched domains. The thermograms exhibited by ternary sphingomyelin/ganglioside/cholesterol mixtures in variable proportions (up to 20% molar GM1 or Chol) displayed, on increasing the content of either the sterol or the ganglioside, features addressable to sphingomyelin/cholesterol (peaks centered at temperature </=40 degrees C, decrease of enthalpy) or to sphingomyelin/GM1 mixtures (peaks centered at a temperature >40 degrees C), respectively. This trend was confirmed by deconvolution analysis, showing that the thermograms are resolvable into components addressable to GM1-enriched and to cholesterol-enriched domains. Taken all together, the results show that the architectural features of sphingomyelin bilayers are strongly dependent on the presence of GM1 ganglioside and cholesterol, whose presence is leading to the formation of separate, GM1-enriched and cholesterol-enriched distinct domains. Ganglioside-sphingomyelin and sphingomyelin-cholesterol, together with mutual ganglioside-ganglioside, interactions could contribute to maintain a network of bonds extending to proteins, forming specialized membrane domains, such as caveolae, or others, whose experimental clues are the glycolipid-enriched detergent-insoluble fractions that can be isolated from cell membranes.

Animals↗

PubMed1996

Characterization of biotinylated liposomes sensitive to temperature and pH: new tools for anti-cancer drug delivery.

We describe a liposome formulation characterized by sensitivity to concurrent and small temperature and pH changes. Liposome permeability was assessed by monitoring the release of entrapped carboxyfluorescein (CF), using a fluorescence dequenching technique. The thermotropic behavior of the liposomes was investigated by differential scanning calorimetry. After 2 h at 37 degrees C in fetal calf serum, liposomes composed of a mixture of dipalmitoyl phosphatidylcholine/cholesterol/GM1 ganglioside/biotinoyl-dipalmitoyl phosphatidylethanolamine (100:20:6:0.25 molar ratio) released 8% CF at pH 7.4 and 12% CF at pH 6.7. At 41 degrees C the leakage was 72% at pH 7.4 and almost complete (99%) at pH 6.7. The pH and temperature sensitivity, with maximal release when the two circumstances occurred simultaneously, was confirmed by entrapping calcein or [14C]glucose. The reasons for the bilayer sensitivity and the conditions for in vivo drug delivery are discussed.

Antineoplastic Agents↗

PubMed1996

Dependence of rat liver CMP-N-acetylneuraminate:GM1 sialyltransferase (SAT IV) activity on the ceramide composition of GM1 ganglioside.

The dependence of CMP-N-acetylneuraminate:GM1 sialyltransferase (SAT IV) activity of rat liver Golgi apparatus on GM1 ganglioside ceramide composition was evaluated. SAT IV activity was assayed on GM1 molecular species carrying homogeneous ceramide moieties containing long chain bases of different length (18 or 20 C atoms) unsaturated or not, linked to 14:0, 16:0, 18:0 or 22:0 fatty acids. The results obtained in the presence of the detergent Triton CF-54, when enzyme and substrate are presumably part of the same supramolecular structure, show that either the long chain base or the fatty acid composition can affect enzyme activity. This feature was not displayed when GM1 was embedded in dipalmitoylphosphatidylcholine vesicles in the absence of detergent. Under the latter conditions, the enzyme was not sensitive to the lipid composition of GM1 but to the ganglioside/phospholipid ratio in the vesicles. These results indicate for the first time that SAT IV is affected by the lipid composition of the substrate and strengthen the hypothesis that glycosyltranferases may contribute to control the cellular glycosphingolipid ceramide pattern.

Animals↗

PubMed1995

A photo-reactive derivative of ganglioside GM1 specifically cross-links VIP21-caveolin on the cell surface.

Previous studies have shown that sphingolipids may be enriched in caveolae, plasmalemmal invaginations implicated in endocytosis and signal transduction. We synthesised a radiolabeled derivative of ganglioside GM1 bearing a photo-reactive cross-linker at the end of its acyl chain. After insertion in the plasma membrane of cultured A431 or MDCK cells and photoactivation, the main protein cross-linked by the GM1 derivative was VIP21-caveolin, an essential structural component of caveolae. This result shows close proximity between GM1 molecules and VIP21-caveolin in the caveolar membrane and strongly implicates sphingolipid segregation in the biogenesis of caveolae.

Animals↗