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Biomedical subjects

L Corazzi

Publications and source records attributed to L Corazzi.

At least 37 records · Page 2Linked to original sources
PubMed1989

Effect of pyridoxal 5'-phosphate and valproic acid on phospholipid synthesis in neuroblastoma NA.

Phospholipid metabolism in neuroblastoma cells in monolayer culture after acute exposure to pyridoxal phosphate (PLP) has been studied. (a) A strong depression of the rate of biosynthesis of cellular phospholipids from labeled choline and ethanolamine, is demonstrated in neuroblastoma cells grown in culture media containing PLP. (b) Valproic acid reverses the effect of PLP on ethanolamine and choline incorporation into cell lipid. Other anticonvulsants (clonazepam, diazepam, carbamazepine, diphenylhydantoin and ethosuximide) have little or no effect on reversing the inhibition of lipid synthesis produced by PLP. (c) PLP decreases the cellular uptake of choline. This effect might be responsible for the decreased lipid synthesis and is partially reversed by valproic acid. (d) The energy charge of the cell is not affected by either PLP or valproic acid, but it is diminished by the two compounds together. (e) The degradation of choline lipids is decreased by PLP and valproic acid. The hydrolysis of phosphocholine and the outflow of choline from cultured cells is also affected by the drugs. Variations of ethanolamine and choline transport should not be due to any effects of PLP or valproic acid on the lipid phase of the membranes since these molecules have no effect on the permeability of liposomes. (f) It is concluded that ethanolamine and choline lipid metabolism in cultured neuroblastoma cells is influenced by PLP and/or valproic acid, probably through a mechanism involving the transport of precursors across the membrane, although other mechanisms cannot be ruled out.

Energy Metabolism↗
PubMed1989

Fusion of liposomes and rat brain microsomes examined by two assays.

Liposomes are prepared from rat brain microsomal lipid and loaded with either Tb3+ or dipicolinic acid (DPA) to test fusion with the Tb-DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R18) to test fusion with the R18 assay. The addition of either Ca2+ or Mg2+ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg2+ is similar to that elicited by Ca2+ if assessed with R18, but much higher if determined by Tb-DPA. The Ca2(+)-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internal to vesicles. The contrary is true for the Mg2(+)-dependent fluorescence. Rat brain microsomes can be disrupted by adding octylgucoside and reconstituted by removing it by dialysis. We use this procedure to load microsomes with DPA. This allows the use of the Tb-DPA assay for testing the fusion of rat brain microsomes. Reconstituted microsomes fuse with liposomes. This fusion has characteristics similar to those of liposome-liposome fusion. However, no microsome-microsome fusion could be detected with either method. The two methods give different results, owing to the chemical properties of the assays. Indeed Tb-DPA implies the retention of vesicle content, whereas this is not required by the R18 assay.

Animals↗
PubMed1989

Effect of subconvulsive doses of bicuculline on the incorporation of radioactive precursors into glycerolipids in rat brain areas.

Bicuculline (either 25 mumol or 12.5 mumol/kg body wt) was administered to rats by intraperitoneal route. Animals treated with 25 mumol/kg experienced convulsions, whereas those receiving 12.5 mumol/kg did not. Controls received saline instead of the drug. Radioactive precursors [2-3H] glycerol and/or [1,2 14C] ethanolamine were injected into cerebral ventriculi simultaneously with bicuculline and the rats were killed 12 min afterwards. Their brains were dissected by hand into four parts (cerebellum, brain stem, hippocampus, cerebral cortex) and the labeling of lipid classes determined after extraction and separation. Although glycerol was incorporated into lipid better than ethanolamine in all areas, the fate of the injected radioactive precursors varied according both to area and treatment. The lowest uptake of radioactivity was in the cerebral cortex and the highest in the brain stem and hippocampus. Moreover, the administration of bicuculline influenced the distribution of radioactivity among lipid classes; these variations, however, were not dependent on the administered doses of bicuculline. We conclude that the effects on glycerolipid metabolism observed in convulsing animals are due to several causes including alterations of systemic parameters (hypertension, hypoxia, etc.). The distribution of glycerol label between phospholipid and neutral lipid is proposed as a biochemical model for the study of convulsive and subconvulsive states.

Animals↗
PubMed1988

The incorporation of intracranially injected glycerol into brain glycerides of young rats born to normal and alcohol-fed mothers.

Growth alters the ability of rat brain to incorporate [2-3H]glycerol into glycerides; indeed, 12 min after the intracranial administration of the precursor, diglyceride becomes more radioactive in newborn than in 19-day-old brain, the reverse being true for total glycerophospholipid and triglyceride. The ratio between the labeling of phospholipid and that of neutral lipid in the experimental conditions described in this paper is proposed as a marker of brain maturity. The distribution of labeling among phospholipid classes also varies with age, and the increase of labeling in total phospholipid occurring with increasing age is almost entirely due to phosphatidylethanolamine and phosphatidylcholine. The metabolism of myelin lipids might be responsible for these age-dependent variations. The administration of ethanol to dams during pregnancy and lactation alters the distribution of the label among neutral glycerolipid and total glycerophospholipid in an age-dependent manner. The labeling distribution among phospholipid classes is also affected.

Aging↗
PubMed1988

Incorporation of glycerol and ethanolamine into glycerophospholipid in rat brain areas during bicuculline-induced convulsive seizures.

The effect of bicuculline-induced convulsive seizures on lipid metabolism has been studied in four brain areas (cerebellum, cerebral cortex, hippocampus, and brainstem) using [2-3H]glycerol and [1,2-14C]ethanolamine as radioactive lipid precursors administered simultaneously with bicuculline. Twelve minutes after the administration, the uptake of radioactivity depended both on brain area and treatment, being generally higher in convulsing rats. The uptake of glycerol was influenced to a larger extent than that of ethanolamine and increased during convulsions, but its incorporation into lipids did not. In contrast, the amount of ethanolamine incorporated into lipids increased during bicuculline-induced seizures. The difference in behavior of glycerol and of ethanolamine is also indicated by the decrease of the 3H/14C ratio of phosphatidyl-ethanolamine in various brain areas during convulsions. It is, therefore, evident that the metabolism of the two precursors is affected differently by seizures.

Animals↗
PubMed1987

Factors affecting the reaggregation of rat brain microsomes solubilized with octyl glucoside and their relationship with the base-exchange activity of reaggregates.

Rat brain microsomal membranes disaggregated by exposure to octyl glucoside were recovered by centrifugation after dialytic removal of the detergent. The composition of the dialysis medium (divalent cations, pH) was important to this effect; indeed, the reaggregation process which occurred during the dialytic step required the presence of either Ca2+ or Mg2+ and a slightly acidic pH. The lipid protein/ratio and choline and ethanolamine base-exchange of recovered particles depended on the conditions of dialysis although their lipid composition did not. The lipid composition of membranes was also varied by adding PE or PC to octyl glucoside-microsome suspensions. This treatment produced reaggregates possessing a low content of cholesterol and varying PC/PE ratios. Both choline and ethanolamine base-exchange activities were related to this parameter.

Animals↗
PubMed1987

Glycerol incorporation into brain lipids in rat pups born to ethanol-intoxicated dams.

The incorporation of labeled glycerol into glycerolipid of rat brain was influenced by the age of the animal (2-month-old vs. newborn); indeed, 12 min after the administration, diglyceride was the most heavily labeled glycerolipid in the newborn brain, whereas the labeling of glycerophospholipid was highest in the adult. Various amounts of ethanol (0% to 36% of total energy intake) were administered to pregnant female rats, and the brains of their pups were examined for the ability to incorporate labeled glycerol into glycerolipid. The radioactivity incorporated into lipid diminished with increasing the amounts of alcohol consumed. The labeling pattern of lipid classes was also influenced; indeed, the radioactivity of diglyceride decreased markedly, whereas that of triglyceride and glycerophospholipid was affected to a lower degree. The distribution of radioactivity among different phospholipids also varied with age; on a percent basis, phosphatidylcholine was labeled less and phosphatidylinositol was labeled more in the newborn than in the adult. Ethanol influenced the pattern of glycerophospholipid labeling, increasing the radioactivity of phosphatidylserine and decreasing that of phosphatidylinositol.

Age Factors↗
PubMed1987

Cerebellar metabolism of phosphatidylethanolamine and its water-soluble precursors during bicuculline-induced convulsive seizures.

The incorporation of labeled ethanolamine into phosphatidylethanolamine (PE) and its water-soluble precursors, phosphoethanolamine and CDP-ethanolamine, is measured in rat cerebella during the course of bicuculline-induced convulsive seizures. The labeling of CDP-ethanolamine and phosphoethanolamine diminishes 6 min after the administration of both bicuculline and radioactive ethanolamine whereas that of PE is unaffected in these conditions. Time is very important to this effect; indeed, no differences of the labeling of PE water-soluble precursors can be found in rat cerebellum of normal and convulsing animals 12 min after the administration. The cerebellar pool of CDP-ethanolamine doubles after 6 min of convulsions, which means that unlabeled CDP-ethanolamine forms from a non-radioactive source, such as lipid, may be through the reversal of the ethanolamine phosphotransferase reaction. This effect disappears 12 min after the injection of the convulsant.

Animals↗
PubMed1986

The reaggregation of rat brain microsomal membranes after the treatment with octyl-beta-D-glucopyranoside. A study on ethanolamine base-exchange.

The ethanolamine base-exchange activity of rat brain microsomes has been studied after treating the membranes with the non-ionic detergent n-octyl-beta-D-glucopyranoside. The detergent could solubilize membrane lipid and protein. The concentrations of the detergent and of membrane protein were both important for this effect. The presence of disaggregating concentrations of octylglucopyranoside in the base-exchange incubation mixture strongly inhibited the incorporation of radioactive ethanolamine into lipid; however, the removal of the detergent through dialytic procedures before assaying the base-exchange reaction restored the enzymic activity almost completely. As shown by exposing the membranes to trinitrobenzenesulfonic acid (TNBS), the phosphatidylethanolamine (PE) which was newly synthesized by base-exchange was also compartmented in the microsomal membrane. The treatment with the detergent after the base-exchange reaction abolished the compartmentation of the newly synthesized lipid. However, if microsomes were solubilized and the detergent was removed by dialysis before the assay of base-exchange, the reassembly of membranes occurred with a recovery of the compartmentation of the newly synthesized PE. The presence of Ca2+ in the dialytic medium was important for the preservation of base-exchange activity, probably affecting the reassembly of membrane components.

Animals↗
PubMed1986

Cerebellar metabolism of phosphatidylcholine and its hydrosoluble precursors during bicuculline-induced convulsive seizures.

The cerebellar incorporation of labeled choline into phosphatidylcholine (PC) and its hydrosoluble choline-containing precursors has been examined during the course of bicuculline-induced convulsive seizures. The labeling of phosphocholine and of PC diminished in these conditions whereas that of cytidine-5'-diphosphate choline (CDP-choline) was practically unaffected. Moreover, the cerebellar pools of phosphocholine and CDP-choline increased by 75-100% after 6 min of convulsions; these compounds were formed from lipid through the action of phospholipases or through the reverse action of choline phosphotransferase. From the data reported in this paper it should also be inferred that the cytidylyltransferase reaction was activated. It is therefore concluded that the cerebellar metabolism of PC and its precursors was affected in various ways by the bicuculline-induced convulsive seizures.

Animals↗
PubMed1986

Compartmentation of newly synthesized phosphatidylethanolamine in rat brain microsomes.

The compartmentation of the phosphatidylethanolamine newly synthesized in brain microsomes in vitro either by base exchange or net synthesis has been studied, using difluorodinitrobenzene as a chemical probe. The experimental results demonstrate that in rat brain microsomes the phosphatidylethanolamine molecules synthesized by base exchange and the bulk membrane lipid belong to different pools. Ca2+ bound to microsomes seems to be involved in the maintenance of the compartmentation of phosphatidylethanolamine. In the presence of Ca2+ the newly synthesized phosphatidylethanolamine molecules react with difluorodinitrobenzene as though they are organized in clusters. After biosynthesis in vivo or in vitro through the cytidine pathway, the compartmentation of the newly formed phosphatidylethanolamine appears less marked than after the synthesis through base exchange.

Animals↗
PubMed1986

Ethanolamine base-exchange reaction in rat brain microsomal subfractions.

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.

Animals↗
PubMed1985

Effect of various drugs producing convulsive seizures on rat brain glycerolipid metabolism.

Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridoxal phosphate, bicuculline, penicillin and ouabain). Glycerolipid metabolism was studied after the intraventricular injection of [2-3H]glycerol, which was incorporated into rat brain glycerides. The percentage of total lipid label found in each lipid class (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidic acid, PA; phosphatidylinositol, PI; diacylglycerol (+ monoacylglycerol), DG and triacylglycerol, TG) depended on the time elapsed from the injection of the labeled precursor. The percent of total lipid radioactivity as PE and PC increased with time (3-60 min), whereas the opposite was true for the radioactivity of DG and PA. The radioactivity of other lipid classes did not appreciably vary between 3 and 60 min from the injection of the labeled glycerol. The intraventricular administration of pyridoxal phosphate together with labeled glycerol decreased the percent of lipid radioactivity as PE and increased that as DG. This 'lipid effect' was detected also after the administration of other convulsants, such as ouabain and penicillin. The intraperitoneal administration of bicuculline affected lipid metabolism in cerebellum.

Animals↗
PubMed1985

Sidedness of phosphatidylcholine-synthesizing enzymes in rat brain microsomal vesicles.

The sidedness of CDP-choline:1,2-diradylglycerol choline phosphotransferase (EC 2.7.8.2) and of the choline base-exchange activity has been studied in rat brain microsomal vesicles. Proteases (trypsin and pronase) and mercury-dextran have been used as reagents for membrane surface components. All of them could inactivate both enzymes to a good extent, without affecting the morphology or the permeability to sucrose of the vesicles. It is therefore concluded that CDP-choline:1,2-diradylglycerol choline phosphotransferase and the choline base-exchange activity are localized on the outer surface of rat brain microsomal vesicles.

Animals↗
PubMed1984

The effect of pyridoxal phosphate-induced convulsive seizures on rat brain phospholipid metabolism.

The intraventricular injection of pyridoxal phosphate (PLP; 1 mumole/brain) to rats causes convulsive seizures beginning 3 min after injection and lasting for about 20 min. The incorporation of [2-3H] glycerol into rat brain glycerides has been studied to ascertain whether treatment with PLP affects the incorporation of label into various lipid classes. The labeling pattern of glycerides is changed by the administration of PLP. The observed alterations begin a few min after injection, together with the convulsive seizures. 1 h after the injection the pattern of labeling of brain glycerides returns to normal. Different glycerides are differently affected by PLP. This work demonstrates that the labeling of diglyceride increases whereas that of phosphatidylethanolamine decreases following PLP administration.

Animals↗
PubMed1984

The effect of polyunsaturated phosphatidylcholine on lipid synthesis in ethanol-intoxicated cultured rat hepatocytes.

Rat liver cells in primary culture have been used to study the influence of ethanol and of a polyunsaturated phosphatidylcholine preparation from soybean lecithin on glycerol incorporation into lipids. Ethanol decreases the incorporation of glycerol into choline and ethanolamine phosphoglycerides and increases that into diglyceride, either when added to the incubation medium or when administered to rats prior to hepatocyte preparation. The addition of the polyunsaturated lipid material (EPL) is able to counteract the effect of ethanol modifying the labeling of glycerol in favour of phospholipids.

Animals↗