PubMed Health⌕ Search

Biomedical subjects

R Massarelli

Publications and source records attributed to R Massarelli.

At least 37 records · Page 2Linked to original sources
PubMed1994

[Morphological characterization of cell lines of human uveal melanoma].

Several cell lines have been derived from an ocular melanoma obtained from an enucleated patient. Three cell types are observed during the time in culture of all the cell lines under study. Two of them have epithelial and spindle shape respectively. A third cell type, having a spheroidal shape, is formed from spindle cells and may be transformed into epithelial cells upon re-seeding. Further experiments showed that the same cell may change of shape following the cycle: spheroidal-->epithelial-->fusiform-->spheroidal. Scanning microscopy shows the coexistence of the three cell shapes in the same culture and the presence of several filaments and processes protruding at the surface of the cells. Transmission electron microscopy shows that the cell lines, in general, contain melanosomes empty or fairly pigmented and several filaments and microtubules. The presence of melanin may be stimulated by seeding of melanoma cells over a "feeder layer" of fibroblasts.

Humans↗
PubMed1993

Possible involvement of the base exchange enzymes in the phospholipid metabolism in LAN-2 cells.

The incorporation of [14C] choline, [14C] ethanolamine, and [14C] serine by LAN-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no differences between TPA-exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. These results suggest that a choline base exchange enzyme was stimulated in TPA exposed cells. In addition, the enhanced incorporation of serine by TPA into its corresponding phospholipid implies the stimulation of the serine base exchange enzyme which is responsible for phosphatidylserine synthesis in mammals. These observations suggest a

Choline↗
PubMed1993

Activation of phospholipases D and A by amphiphilic cations of cultured LA-N-2 cells is G protein- and protein kinase C-independent.

Several amphiphilic cations such as mepacrine, desipramine, didodecyldimethylamine, chlorpromazine, oleylamine and W-7 activated the phospholipase D (PLD) activity of cultured LA-N-2 cells. These compounds, except for oleylamine, provoked the release of fatty acids, suggesting phospholipase A activation. Melittin, a PLA2 stimulator, caused the robust release of the free fatty acids but was a poor PLD activator. Although PLD could be activated by GTP gamma S, the stimulation by these amphiphilic cations was not abolished by GDP beta S, an inhibitor of G protein function. There was no change in the PLD activation by these amphiphilic cations by DiC8, a PKC activator, or by H-7, a PKC inhibitor or in PKC down-regulated cells.

Amines↗
PubMed1992

Properties of a partially purified phosphodimethylethanolamine methyltransferase from rat brain cytosol.

The presence of cytosolic S-adenosylmethionine-dependent N-methyltransferase(s) activity(ies) capable of converting phosphoethanolamine into phosphocholine has been recently demonstrated in the rat brain. At least two enzymes are involved in the methylation of phosphoethanolamine to phosphocholine and these are separable by ammonium sulphate fractionation. The enzyme catalysing the last step of this methylation process is present in the 50-80% ammonium sulphate fraction. A 220-fold purified enzyme has been obtained with sequentially employed Q-Sepharose fast flow and octyl-Sepharose CL4B column chromatography. The maximum enzyme activity was at pH 9.5. The Km values for S-adenosylmethionine, the methyl donor, and phosphodimethylethanolamine, the methyl acceptor, were 125 microM and 750 microM respectively. This phosphodimethylethanolamine N-methyltransferase was found to be calcium-dependent, with a 4-fold increase in activity at 0.5 mM-CaCl2. S-Adenosylhomocysteine at 0.5 mM caused 100% inhibition of the activity. The effects of various structural analogues on the phosphodimethylethanolamine N-methyltransferase activity were also investigated and these results suggest that the enzyme is specific to the substrate. These results provide evidence for the existence of the pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain cytosol.

Ammonium Sulfate↗
PubMed1992

Oleoylamine and sphingosine stimulation of phosphatidylserine synthesis by LA-N-2 cells is protein kinase C independent.

The presence of sphingosine and oleoylamine in the culture medium of LA-N-2 cells stimulated the incorporation of [3H]serine into its corresponding phospholipid, phosphatidylserine (PtdSer). The optimum stimulation for sphingosine and oleoylamine were 50 microM and 100 microM, respectively. Oleoylamine increased the incorporation of [3H]serine over 6-fold while sphingosine increased the incorporation of [3H]serine over 2.5-fold. The amount of radioactivity found in water-soluble components and in protein was similar to that found with control LA-N-2 cells. The incorporation of [3H]choline and [3H]ethanolamine into their corresponding phospholipids were decreased in the presence of either oleoylamine or sphingosine. A protein kinase C (PKC) activator, DiC8, and a PKC inhibitor, H7, did not influence the enhanced phosphatidylserine formation by sphingosine and oleoylamine. In addition, there were no differences in the stimulatory effect of sphingosine and oleoylamine discernable between PKC down-regulated cells or controls. These observations indicate that this oleoylamine and sphingosine mediated enhanced phosphatidylserine synthesis is PKC-independent.

Amines↗
PubMed1992

Exogenous gangliosides modulate calcium fluxes in cultured neuronal cells.

Previous work from this and other laboratories has shown that the neuritogenic effect due to exogenous gangliosides on primary neurons in culture is accompanied by several morphological and biochemical modifications. The present results indicate that the treatment of these neurons with gangliosides, under the experimental conditions which are known to produce a sprouting effect, inhibited the influx of 45Ca2+ and increased the release of 45Ca2+ from the cells. No significant differences were noted using concentrations of gangliosides (10(-8)-10(-5) M) either below or above the critical micellar concentrations. No apparent specificity was observed among various species of individual sialocompounds (GM1, GD1a). Moreover the presence or absence of fetal calf serum in the culture medium influenced the levels of 45Ca2+ fluxes. This study confirms the hypothesis that gangliosides may be considered as Ca2+ flux modulators in neuronal cells.

Animals↗
PubMed1992

Phorbol esters modulate phospholipid metabolism in a human cholinergic cell line, LA-N-2: a possible role for the base exchange enzymes.

It was shown that in LA-N-2 cells prelabeled with [3H-methyl]choline for 24 hr (Singh et al.: Mol Chem Neuropathol 14:53-66, 1991) the major intracellular and extracellular hydrophilic compound was phosphorylcholine. LA-N-2 cells were labeled with [14C-methyl]choline for 24 hr, harvested, and incubated in Hepes/BSA/saline buffer for varying periods of time. The radioactive compound present in the cytosol and released into Hepes/BSA/saline buffer medium in the presence or absence of TPA was phosphorylcholine. There was a gradual increase in the appearance of radioactivity in the medium and this corresponded to a gradual decline in the radioactivity present in the cytosolic compartment with a statistically significant P value of less than .005. Identical results were obtained with prelabeled cells subsequently incubated with TPA. There was no significant change in the amount of radioactivity associated with lipid suggesting that the phosphorylcholine may be released directly from the cytosolic compartment into the medium rather than originating through a phospholipase-C catalyzed hydrolysis of phosphatidylcholine. This possibility received support from experiments in which cells were electropermeabilized in the presence of radioactive phosphorylcholine. It was found that the introduced [14C]phosphorylcholine was released intact into the incubation medium from the cytosolic compartment. The incorporation of [14C]choline, [14C]ethanolamine, and [14C]serine by LA-N-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no detectable differences between TPA exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine, or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. This effect of TPA in altering the distribution of labeling of the cytosolic and lipid components was not demonstrable with cells grown in the presence of 10(-5) M retinoic acid.

Cells, Cultured↗
PubMed1992

Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line.

Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (ChAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10(-5) M retinoic acid (RA) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a Bmax of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An IC50 of 2.5 x 10(-8) M and 0.9 x 10(-8) M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7 x 10(-2) M and 3 x 10(-2) M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC50:110 microM) thus suggesting the presence of an M3 receptor. Acetylcholine (100 microM) plus eserine (50 microM) and BW284c55 (1 microM), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Modulation of phosphatidylserine homeostasis by amphiphilic cations in a human neuronal cell line, LA-N-2.

The stimulatory effects of sphingosine and oleylamine upon the incorporation of [3H]serine into its corresponding phospholipid, phosphatidylserine, by LA-N-2 cell cultures, have been reported (Singh et al. (1992) FEBS Lett., 296, 166-168). The presence of chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in cultures of these cells provoked increased phosphatidylserine synthesis but decreased protein synthesis in a concentration-dependent fashion. Several other amphiphilic cations, including dimethyldidodecylammonium bromide, verapamil, desipramine, amitriptyline, and mepacrine also stimulated phosphatidylserine synthesis in a dose-dependent manner. These amphiphilic cations reduced the conversion of phosphatidylserine to phosphatidylethanolamine. The observed stimulation of phosphatidylserine synthesis by amphiphilic cations was unaltered in protein kinase C (PKC) down-regulated cells and in retinoic acid (RA)-induced differentiated cells. In contrast, bis(2-ethylhexyl)hydrogen phosphate, an amphiphilic anion, strongly inhibited the incorporation of [3H]serine into phosphatidylserine. These observations demonstrate that phosphatidylserine synthesis in LA-N-2 cells can be modulated by amphiphilic cations and anions. It is suggested that the amphiphilic cations cause the ethanolamine moiety of membrane bound phosphatidylethanolamine to become more accessible to the juxtaposed serine base exchange enzyme.

Anions↗
PubMed1991

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures.

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10(-5)M and 10(-8)M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [3H] and [35S] methionine and [3H]ethanolamine as precursors showed an increased methylation of [3H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [3H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [3H]- and [35S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased 3H- and 35S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [3H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-3H] label after incubation of neurons with [3H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.

Animals↗
PubMed1991

Stimulation of mono- and diacylglycerol lipase activities by gangliosides in chicken neuronal cultures.

Chicken neurons in culture display measurable activities of mono- and diacylglycerol lipases. Treatment of chicken neuronal cultures with gangliosides (10(-8)M to 10(-5)M) resulted in a time and dose dependent increase in monoacylglycerol lipase activity. The diacylglycerol lipase showed significant increase in specific activity before that of monoacylglycerol lipase. The increase was observed only up to 24 hours and no differences between diacylglycerol lipase activity of control and ganglioside treated cells were observed after 48 hours. The data indicate that the treatment of neurons with exogenous gangliosides affect the diglyceride metabolism in stimulating not only the enzymes catalyzing their production but also those involved in their catabolism.

Animals↗
PubMed1991

The metabolic fate of [3H-methyl]choline in cultured human neuroblastoma cell lines, LA-N-1 and LA-N-2.

The conversion of choline in cultures of the human neuroblastoma cell lines, LA-N-1 and LA-N-2 cells, was investigated in order to identify potential precursors in acetylcholine (AcCho) synthesis. LA-N-1, a catecholaminergic and LA-N-2, a cholinergic, cell line were incubated with [3H-methyl]choline (Cho) for varying periods of time up to 72 h. The radioactivity present in lipids and water-soluble metabolites increased linearly up to 24 h in both cell lines. Approximately 20% of the radioactivity associated with the water-soluble metabolites in both control (untreated) and retinoic acid-induced differentiated (RA-treated cells) LA-N-2 cells was present as Cho and AcCho. There was no detectable AcCho in the catecholaminergic cell line, LA-N-1. The untreated and RA-treated LA-N-1 and LA-N-2 cells were labeled for 24 h with [3H-methyl]Cho, followed by a chase in growth medium containing 100 microM unlabeled choline. The distribution of radioactivity in the LA-N-2 cells was 6-10% of AcCho, 84-89% as phosphocholine (PCho), 1-3% as glycerophosphocholine (GroPCho), and 2-4% as Cho. The distribution of radioactivity in the LA-N-1 cells was similar except for the absence of AcCho. The distribution of radioactivity in the culture medium of LA-N-1 cells was 70-80% as Cho, 20-30% as PCho, and 1-3% as GroPCho. In contrast, the radioactivity was equally distributed between Cho (50%) and PCho (50%), with only 1-3% as GroPCho in the medium of LA-N-2 cells.

Acetylcholine↗
PubMed1991

Effect of monomethylethanolamine, dimethylethanolamine, gangliosides, isoproterenol, and 2-hydroxyethylhydrazine on the conversion of ethanolamine to methylated products by cultured chick brain neurons.

The sequential methylation of ethanolamine and its phosphorylated derivatives has been studied with chick neurons in culture in the presence of several pharmacological agents. Incubation with [3H]ethanolamine in the presence of monomethylethanolamine and dimethylethanolamine indicated that in these neurons the preferential conversion to choline-containing compounds is via the methylation of phosphorylethanolamine. The possibility that there are two separate enzymes, i.e., one responsible for the methylation of water-soluble ethanolamine-containing compounds and another for the ethanolamine phospholipids, was examined with agents believed to influence these conversions. Incubation of neurons in the presence of a mixture of exogenous gangliosides at 10(-8) M and 10(-5) M concentrations showed that these neuritogenic compounds stimulate the methylation of phosphatidylethanolamine and decrease that of phosphorylethanolamine. The inhibitor of phosphatidylethanolamine methyltransferase (EC 2.1.1.17), 2-hydroxyethylhydrazine, decreased the conversion of phosphatidylethanolamine to phosphatidylcholine and increased that of phosphorylethanolamine to phosphorylcholine. The possible effects of adrenergic stimulation were studied by the incubation of neurons with isoproterenol at 10(-6) M and 10(-5) M concentrations. There was a reduction of phosphorylethanolamine methylation and a stimulation of that of phosphatidylethanolamine, and these effects were counteracted by the presence of 5 x 10(-5) M propranolol.

Animals↗
PubMed1990

A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine.

It had been previously speculated upon but never proved that the methylation of phosphorylethanolamine could contribute to the production of choline containing compounds. However, experimental evidence obtained with neuronal cultures was interpreted as showing that the stepwise methylation of phosphobases may be an important route for this biosynthesis. We demonstrate that cytosolic fraction from rat brain possesses a N-methyltransferase activity capable of methylating phosphorylethanolamine and its mono- and dimethyl-derivatives into phosphorylcholine. The level of activity detectable in rat liver cytosol is only 18% of that found in the brain cytosol.

Animals↗
PubMed1990

5 alpha-reductase activity in isolated and cultured neuronal and glial cells of the rat.

The distribution of the 5 alpha-reductase, the enzyme which converts testosterone into its 'active' metabolite dihydrotestosterone (DHT), has been studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation and in neurons and glial cells grown in cultures. Purity of cellular preparations was examined by electron and light microscopy. Purified neurons, astrocytes and oligodendrocytes, obtained from the brain of adult male rats, are all able to form DHT from testosterone and consequently possess a 5 alpha-reductase activity. Among the 3 cell types studied, neurons appear to be more active than oligodendrocytes and astrocytes. Moreover, between the two population of glial cells, the oligodendrocytes seem to possess a slightly higher enzymatic activity than that present in the astrocytes. Neurons appeared more active in metabolizing testosterone than glial cells also in cell culture experiments. It is presently believed that the 5 alpha-reduction of testosterone to DHT provides one of the mechanisms through which the hormone becomes effective in the CNS. This is supported by the present findings, which indicate that neurons are the cell population in which the 5 alpha-reductase is more concentrated. However, the presence of a considerable 5 alpha-reductase activity in glial cells indicates that also non-neuronal cells might participate in androgen-mediated events occurring in the brain.

Animals↗
PubMed1990

In vitro synthesis and transbilayer movement of phosphatidylethanolamine molecules labelled with different fatty acids in chick brain microsomes.

The transbilayer fatty acid distribution of diacylglycerophosphoethanolamine and the translocation of newly synthesized phosphatidylethanolamine molecules labelled with different fatty acids has been investigated in chick brain microsomes using trinitrobenzensulfonic acid. The determination of the fatty acid composition of diacylglycerophosphoethanolamine in both the outer and the inner leaflet of the microsomal vesicles revealed a similar distribution indicating that both leaflets share the same molecular species. The in vitro incorporation of radioactive fatty acids (16:0, 18:1 and 20:4(n-6] into ethanolamine phospholipids, known to be catalyzed by the lyosphosphatidylethanolamine acyl transferase, showed that the radioactive diacylglycerophosphoethanolamine molecules appeared first in the outer leaflet and were thereafter transferred to the inner leaflet. The apparent rate of translocation of the newly synthesized ethanolamine phospholipid molecules was the highest for those labelled with 16:0 and the lowest for those labelled with 20:4(n-6). The results indicate that the active site of the acyl-CoA:lysophosphatidylethanolamine acyltransferases is located on the outer leaflet of the microsomal vesicles and that the different newly synthesized molecular species of diacylglycerophosphoethanolamine may be translocated from the outer to the inner leaflet at different rates.

Animals↗
PubMed1990

Enzymatic activities during differentiation of the human neuroblastoma cells, LA-N-1 and LA-N-2.

The presence of 10(-5) M retinoic acid (RA) in the culture medium of LA-N-1, a catecholaminergic cell line, and LA-N-2, a cholinergic cell line, enhanced their morphological differentiation. Tyrosine hydroxylase (TH) activity of the LA-N-1 cells was increased in the RA-treated cells compared with control cultures at day 4 and remained elevated. Choline acetyltransferase (ChAT) activity in the LA-N-2 cells gradually increased until 8 days in vitro (DIV) both in the untreated control and the RA treated cultures. This activity in control and treated cells decreased gradually to a constant level of activity. The ChAT activity at 8 DIV of RA-treated LA-N-2 cells was increased 2.1-fold (P less than 0.001) as compared to the control cultures. This increase in ChAT activity was accompanied by a 73% decrease of acetylcholinesterase (AChE) activity in LA-N-2 cells by 8 DIV. AChE activity of LA-N-1 cells was unchanged during the time course of the experiment. Phospholipase-A2 (PL-A2) activity in RA-treated LA-N-2 cells was increased at day 4 as compared with the control cultures. There were no differences observed in phospholipase-D (PL-D), choline kinase and GPC-phosphodiesterases activities in RA-treated and -untreated LA-N-1 and LA-N-2 cells.

Acetylcholinesterase↗