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J M Pasquini

Publications and source records attributed to J M Pasquini.

At least 55 records · Page 3Linked to original sources
PubMed1985

Bradykinin stimulates phospholipase C in rat renal medullary slices.

Rat renal medullary slices prelabeled with [14C]arachidonic acid generate [14C]diacylglycerol within 1 min of exposure to bradykinin action. Production of [14C]diacylglycerol is transient. 2 min after the addition of bradykinin, the levels of metabolite reach the maximum, but decrease thereafter. Simultaneously, bradykinin induces a parallel decrease of the radioactivity in phosphatidylinositol. No degradation of other phospholipids is observed, and triacylglycerol is not affected. The degradation of [14C]phosphatidylinositol to [14C]diacylglycerol indicated the presence of phospholipase C activity. Preincubation of prelabeled slices with 2 mM dibutyryl cyclic AMP prevents both the generation of diacylglycerol and the degradation of phosphatidylinositol. Neither mepacrine nor indomethacin block diacylglycerol production and phosphatidylinositol breakdown. We conclude that, when rat renal medullary slices are stimulated with bradykinin, phosphatidylinositol-specific phospholipase C is activated.

Animals↗
PubMed1985

Deacylation of myelin proteolipid protein in organic solvents.

A procedure has been developed for the deacylation of the hydrophobic, myelin proteolipid apoprotein using hydroxylamine in an alkaline organic solvent medium. Complete removal of covalently bound fatty acids was obtained after 4 hr of treatment. After deacylation, no changes could be detected in the electrophoretic pattern or in the number of free sulfhydryl groups. The deacylated apoprotein remains soluble in chloroform-methanol mixtures and is suitable for further physicochemical characterization.

Acylation↗
PubMed1985

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin.

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [35S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.

Animals↗
PubMed1985

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: effect of malnutrition.

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [3H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18-21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain a normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.

Age Factors↗
PubMed1985

Electrophoretic analysis of myelin proteolipid protein and its deacylated form.

Myelin proteolipid protein is known to contain covalently bound fatty acid. To determine the contribution of the fatty acid to the multiple bands observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretic parameters of the proteolipid protein were compared with those of the deacylated form. The relative mobility and proportion of each band, as well as the retardation coefficient and free electrophoretic mobility, were not altered by removal of the fatty acid moiety. Furthermore, the acylated and deacylated forms bound the same amounts of sodium dodecyl sulfate. These data demonstrate that the presence of covalently bound fatty acids does not account for the electrophoretic heterogeneity of the proteolipid.

Acylation↗
PubMed1984

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system.

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [3H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDS-PAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulse-chase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.

Animals↗
PubMed1984

Mechanisms of transport and assembly of myelin proteins.

The present study was carried out in order to obtain further information regarding the mechanism of transport and assembly of myelin proteins in different subcellular fractions isolated from brain slices incubated in vitro with radioactive amino acids under different experimental conditions. It was found that proteolipid protein (PLP) showed a lag in the entry into the myelin membrane, while basic and Wolfgram proteins appeared to be inserted in this structure immediately after their synthesis. Addition of 500 microM colchicine to the incubation medium blocked the transport of PLP, while the entry of the other proteins was not affected. Pulse-chase experiments using cycloheximide suggest that a precursor-product relationship between microsomes, fraction SN4 and myelin exists only for PLP. The results obtained allow us to draw the following conclusions: The delay in the entry of PLP into myelin membrane is probably due to the time required for its transport towards the final site of assembly; the microtubular network of the oligodendroglial cell is directly involved in the transport of PLP; basic and probably Wolfgram proteins follow a route which clearly differs from that of PLP; delivery of myelin proteins from the site of synthesis towards their site of deposition depends, at least, on two different mechanisms of intracellular transport.

Animals↗
PubMed1982

An electrophoretic analysis of proteolipids from different rat brain subcellular fractions.

Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of the delipidized proteins, in the presence or absence of 8 M urea, was carried out with all fractions. The distribution of the various types of proteolipid proteins was studied and their molecular weight calculated by the Ferguson relationship. Several bands of proteolipid proteins were found in the five membrane fractions analyzed. Some of them, such as the 17.5 K and 37 K components were very prominent in mitochondria and synaptosomes. The 30 K component was found in myelin-derived membranes and in microsomes, while the 20 K and 25 K proteolipid proteins were present in all subcellular fractions. The 30 K component (proteolipid protein (PLP)), typical of the purified myelin membranes, showed a similar distribution to that of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (EC 3.1.4.37) activity, while the other major proteolipid protein present in all subcellular fractions (25 K) did not show such parallelism, indicating that it might not be an exclusive component of myelin. The electrophoretic pattern of microsomal proteolipid proteins did not show the high molecular weight components (aggregates of PLP) which are found in myelin. Furthermore, the 30 K component showed a smaller Y0 value than that of the 30 K found in myelin. Thus the presence of 30 K proteolipid protein in microsomes should not be considered as being due to myelin contamination.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1982

Rapid purification of proteolipids from rat brain subcellular fractions by chromatography on a lipophilic dextran gel.

Proteolipids from adult rat brain subcellular fractions were purified by a one-step procedure involving chromatography through Sephadex LH-60 eluted with an acidified chloroform-methanol mixture. The protein peak was eluted with the void volume and was free of adventitious lipids. The degree of purification was similar to that attained with the neutral-acidified chloroform-methanol dialysis method with the advantage that this new procedure can be carried out in only 3 h, with a recovery of proteins of 95-100%. Samples containing different lipid/protein ratios passed through the gel gave similar elution profiles. When labeled amino acids or palmitic acid were added to myelin total lipid extracts, no radioactivity was eluted with the protein, indicating that the proteolipid apoproteins purified by this method do not adsorb hydrophobic low-molecular-weight compounds.

Animals↗
PubMed1982

Effect of thyroid dysfunction upon phospholipid composition and CDP-choline incorporation in mitochondria and microsomal fraction isolated from liver and brain of suckling rats.

The phospholipid composition and the in vitro incorporation of radioactive CDP-choline into phosphatidylcholine was studied in mitochondria and microsomal fraction obtained from liver and brain of 20 day old hyperthyroid or hypothyroid rats. The chemical composition of the subcellular membranes isolated from brain differed markedly in both conditions. In hyperthyroidism the microsomal fraction was slightly affected while the mitochondria were also affected, but not as severely as in hypothyroidism, in which the microsomal fraction showed no alterations. The incorporation of the radioactive precursor into brain mitochondria isolated from hyperthyroid rats was markedly decreased, while no changes were observed in microsomes. However, incorporation into brain microsomal fraction obtained from hypothyroid rats was increased, while no changes were observed in mitochondria. similar results were obtained in the studies performed with liver subcellular membranes from hyperthyroid animals while no changes were found in those from hypothyroid rats. Our results indicate that both experimental conditions affect in a different way the structure and function of brain mitochondria and microsomal fractions. They also give further support to our hypothesis that mitochondria have a certain degree of autonomy for the synthesis of phosphatidylcholine.

Animals↗
PubMed1982

Differential effect of colchicine upon the entry of proteins into myelin and myelin related membranes.

Brain slices from 20 day old rats were incubated with radioactive aminoacids in the presence and absence of 500 microM colchicine and the appearance of labeled proteins in myelin and in a myelin-like fraction (SN4 fraction) was measured. In the presence of the inhibitor, the entry of proteolipid proteins was decreased to 55% in myelin and to 45% in SN4 fraction with reference to control values while the entry of basic proteins and other minor protein components was unaffected in both fractions. The synthesis of proteolipid proteins was not affected by the presence of colchicine; moreover, a slight accumulation of these proteins was observed in microsomes. The results suggest that the microtubular system is involved in the transport of proteolipid proteins from their site of synthesis to their site of deposition and that the various types of myelin proteins follow different transport routes to enter into this special type of membrane.

Animals↗
PubMed1981

Neonatal hypothyroidism and early undernutrition in the rat: defective maturation of structural membrane components in the central nervous system.

The action of neonatal hypothyroidism and early undernutrition on the lipid and protein composition, as well as, on the activity of 2'3' cyclic nucleotide 3' phosphohydrolase was studied in different subcellular fractions isolated from 20 day old hypothyroid and undernourished rats. Based on protein content, a marked decrease (70%) in the recovery of myelin was observed in both experimental conditions. The lipid composition of myelin in both groups was, however, different; while cholesterol, total phospholipids, and total galactolipids decreased in a similar fashion in the two situations; sulfatides and plasmalogens were much more affected in hypothyroid rats.

Animals↗
PubMed1980

Differential effect of L-thyroxine on phospholipid biosynthesis in mitochondria and microsomal fraction.

1. The action of L-thyroxine on the incorporation of radioactive choline or CDP-choline into phosphatidylcholine in vitro was explored in liver and brain microsomal fraction and mitochondria obtained from young adult rats. 2. In liver mitochondria isolated from animals treated with L-thyroxine (40 mg/kg body wt. during 6 days), the incorporation of both radioactive precursors into phosphatidylcholine was significantly decreased compared with normal controls, whereas in the total homogenate and in the microsomal fraction the incorporation was similar in the experimental and control groups. In subcellular fractions isolated from brain, the incorporation of precursors was similar in L-thyroxine-treated and normal animals. 3. Liver mitochondria isolated from normal animals incubated in vitro with CDP-choline, in the presence of different concentrations of L-thyroxine, showed also a marked decrease in the incorporation of label into phosphatidylcholine, whereas no significant changes were found in the total homogenate and in the microsomal fraction compared with control experiments. 4. The differential effect of L-thyroxine on the incorporation of radioactive precursors into phosphatidylcholine of isolated liver subcellular fractions gives further support to the hypothesis that liver mitochondria can independently synthesize part of their own phospholipids. 5. Possible mechanisms of the action of the hormone at the mitochondrial level are discussed.

Animals↗
PubMed1979

Turnover of phosphatidyl choline and changes in enzymatic activity in cell membranes of the CNS during early myelination.

Subfractions of fraction A, which floats on top on 0.8 M sucrose in the classical density gradient used for the isolation of brain subcellular fractions (A1 corresponding in adult rats to myelin and A2 which corresponds to the myelin-like fraction), were studied in comparison to other brain subcellular fractions in 5 day old rats and at different stages of development, up to 60 days of age. Variations in the enzymatic activity of acetylcholinesterase, non-specific cholinesterase and 2',3'-Cyclic nucleotide phosphohydrolase, changes in lipid and protein composition and turnover of phosphatidyl choline were investigated. Results indicate that fractions A1 and A2 obtained prior to the beginning of myelination could be composed of fragments of the oligodendroglial cell plasma membranes, and that both fractions undergo substantial changes in chemical composition, enzymatic activity and in turnover of phosphatidyl choline during maturation. In vivo experiments at short times, using radioactive choline as a precursor of phosphatidylcholine suggest that membrane fragments isolated in fraction A2 are precursors of those isolated in fraction A1 at all ages.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1979

Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.

A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described. Sphingomyelin species from bovine brain and sheep and pig erythrocytes were resolved into 10-12 separate peaks on a micro -BondaPak C(18) or Nucleosil-5-C(18) reversedphase column with methanol-5 mM potassium phosphate buffer, pH 7.4, 9:1 (v/v) as the solvent. Detection was at 203-205 nm. The sphingomyelin species were primarily resolved due to specific hydrophobic interaction of their fatty acid and sphingoid chains with the alkyl ligand of the stationary phase. The retention time of the sphingomyelin species increased progressively as the number of carbon atoms in the hydrophobic chains increased in the homologous series. The presence of one double bond in the molecule reduced the retention time significantly. Introduction of a second double bond in the fatty acid side chain did not reduce the retention time to the same extent as the first double bond. The presence of a trans double bond in the sphingoid moiety increased the retention time of sphingomyelin more than did a cis double bond in the fatty acid side chain. The differential hydrophobic interaction observed between the ligand of the stationary phase and different alkyl chains of the sphingomyelin species illustrates that reversed-phase HPLC technique can be conveniently used to study the extent of relative hydrophobicity of different types of alkyl chains.-Jungalwala, F. B., V. Hayssen, J. M. Pasquini, and R. H. McCluer. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.

Animals↗