Involvement of phospholipids in polyunsaturated Fatty Acid synthesis in developing soybean cotyledons

Developing soybean (cv. Dare) cotyledons harvested at 30 days after flowering were pulse-labeled with [1-(14)C]oleoyl-CoA. The metabolic interrelation of radiolabeled unsaturated fatty acids between the major glycerolipid classes was determined at various time intervals. At chase time zero, [(14)C]oleic acid accounted for 99.2% of the total glycerolipid radioactivity, and phospholipids contained 92% of the total incorporated radioactivity. With time, phospholipids were metabolized in triacylglycerol biosynthesis and radioactivity was detected in polyunsaturated fatty acids. The hypothesis that phospholipids were metabolic intermediates in polyunsaturated fatty acid biosynthesis was tested by comparing the theoretical and the actual amount of radiolabeled oleic acid that was associated with triacylglycerol as a function of time. The radioactive oleic acid found in triacylglycerol at various intervals was derived from phospholipids via a diacylglycerol intermediate. Assuming no phospholipid desaturation, the potential or theoretical amounts of [(14)C]oleic acid that could be transferred to triacylglycerol from phospholipids was defined by a system of differential equations. The results demonstrated that the decline in [(14)C]oleic acid from phospholipid after long chase intervals was equal to the total amount of radioactive unsaturated fatty acids found in neutral lipids. The difference between the theoretical and actual amounts of [(14)C]oleic acid present in triacylglycerol after long time intervals was equal to the amount of radioactivity present in polyunsaturated fatty acids. Based upon those findings in soybeans, the desaturation of oleic acid associated with phospholipids was highly probable.     

Reduced labeling of brain phosphatidylinositol,triacylglycerols, and diacylglycerols by [1-14C]arachidonic acid after electroconvulsive shock: Potentiation of the effect by adrenergic drugs and comparison with palmitic acid labeling

The effect of electroconvulsive shock on the labeling of phospholipids and neutral lipids in mice brains was examined after intracerebral injection of [1-¹⁴C] arachidonic acid or [1-¹⁴C]palmitic acid. Electroconvulsive shock reduced greatly the removal of radiolabeled arachidonic acid from the free fatty acid pool. At the same time, the incorporation of arachidonic acid was partially inhibited in triacylglycerol, diacylglycerol, and phosphatidylinositol, whereas the incorporation of [1-¹⁴C]palmitic acid was not affected. Pretreatment with desipramine and pargyline potentiated the lipid effect of electroconvulsive shock in neutral glycerides. These electroconvulsive shock-induced changes reflect alterations in the metabolism of intracerebrally injected arachidonic acid, but not of similarly injected palmitic acid. From the available data whether decreased ATP, enzyme inhibition or other factors are involved cannot be ascertained. Moreover, the electroconvulsive shock-enhanced endogenous free arachidonic acid may possibly dilute the injected radiolabeled fatty acid, thus decreasing its availability for arachidonoyl-coenzyme A synthesis. Hence, a partial inhibition of the activation-acylation of these fatty acids, primarily arachidonic acid, also may be involved in the seizure-induced accumulation of free fatty acids in the brain.     

Phospholipid metabolism following fertilization in sea urchin eggs and embryos.

Phospholipid metabolism during early development was examined in the sea urchins Stronglyocentrotus purpuratus and Lytechinus pictus. Transport of ³H-choline was stimulated fivefold following fertilization in both species. However, the actual percent incorporation of labeled precursors into phospholipids from the TCA soluble pool did not change at fertilization. There was a slight increase in transport of ¹⁴C-ethanolamine at fertilization but again there was no change in its percent incorporation into phospholipids. When eggs were preloaded with ³H-choline or ¹⁴C-ethanolamine and fertilized, the eggs or embryos showed similar patterns of incorporation into phospholipids. There was no significant change in the percent phosphorylation of choline in fertilized or unfertilized eggs.An investigation was made of the activity of choline kinase, the first enzyme in the biosynthesis of phosphatidylcholine. This enzyme was found to have similar activities in fertilized and unfertilized eggs using a variety of homogenization media. The activity of choline kinase was found to decrease slightly in activity at fertilization and reach a maximum activity by gastrula.These results indicate that there is no activation of phospholipid synthesis at fertilization of sea urchin eggs. Apparent increased incorporation actually reflects increased transport of precursors and not de novo synthesis.     

Temperature Dependence of Membrane Lipid Composition in Early Blastula Embryos of Lytechinus pictus: Selective Sorting of Phospholipids into Nascent Plasma Membranes

Lytechinus pictus eggs were fertilized and incubated at 10, 16, and 23°C until the early blastula stage of embryonic development. The phospholipid composition of the embryos and control unfertilized eggs remain identical and unchanged as incubating temperatures are varied; thus, neither incubating temperature, fertilization nor membrane assembly affect their total phospholipid composition. This result agrees with metabolic studies by others, using only a single incubation temperature, and indicates that embryonic development to the early blastula stage occurs with little, if any, de novo phospholipid biosynthesis. However, as in all poikilotherms, the phospholipid composition of the nascent plasma membranes varies with the incubation temperature. Thus, until the blastula stage of embryonic development, the lipids of these newly formed plasma membranes are derived from lipid pools within the embryo whose phospholipid composition is static. The variation of plasma membrane composition is primarily reflected in an increase in the phosphatidylethanolamine (PE): phosphatidylcholine (PC) ratio as incubating temperatures decrease; this is achieved by an exchange of PE for PC. Several mechanisms are considered for the specificity of the selective sorting and assembly of these phospholipids into the nascent plasma membranes. Received: 16 March 1999/Revised: 15 May 1999     

Diacylglycerol on lipid metabolism

PURPOSE OF REVIEW: Diacylglycerol is an intermediate product of triacylglycerol hydrolysis and comprises up to 10% of glycerides in plant-derived edible fats and oils. Recent developments in oil chemistry have led to the availability of a novel diacylglycerol oil for clinical studies. Recent research has shown that the oil containing 70% of unusual 1,3- species has metabolic characteristics distinct from those of triacylglycerol of similar fatty acid composition. This review summarizes recent research in humans and experimental animals into the metabolic effects and possible mechanisms of action of this oil. RECENT FINDINGS: Consumption of the oil affects lipid metabolism including lowering of plasma triacylglcerol, decreases postprandial lipemia and reduces body fat mass, compared with triacylglcerol. As the fatty acids of the two oils are similar, the metabolic differences reside in their structural differences. SUMMARY: It is still uncertain whether longer term consumption of the diacylglycerol oil will lead to persistent and consistent reductions in plasma triacylglycerol and body fat. However future studies may demonstrate a role in managing aspects of the metabolic syndrome.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Modification of fatty acid composition of membrane phospholipid in hepatocyte monolayer with n-3, n-6 and n-9 fatty acids and its relationship to triacylglycerol production

The objective of these studies with rat hepatocytes in primary culture was to establish that: (a) membrane phospholipids would become enriched with the specific fatty acid supplemented to the media and (b) hepatocyte monolayer triacylglycerol synthetic rates were dependent on the type of fatty acid enrichment of the membrane phospholipids. Hepatocytes cultured in the absence of media lipid developed a phospholipid fatty acid composition which is indicative of an essential fatty acid deficiency. The extensive rise in 18:1(n - 9) content indicated that delta 9-desaturase was active. The fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the microsomal- and mitochondrial-enriched fractions was highly dependent upon the type of fatty acid supplemented to the medium. Incorporation of fatty acids into phospholipids was rapid, and a new steady-state in fatty acid composition was achieved within approx. 36 h. Changes in the fatty acid composition of these hepatocyte phospholipid subclasses resulting from media supplementation with 18:2/20:4(n-6) or 20:5(n-3) were similar, but not identical, to changes which occurred in vivo as a result of consuming diets rich in 18:2(n-6) or 20:5(n-3). Hepatocyte lipogenesis was highly dependent upon the type of fatty acid supplemented to the medium. Prior conditioning with 16:0 increased triacylglycerol synthesis and secretion. Secretion of triacylglycerol was reduced by polyenoic fatty acid enrichment with 20:5(n-3) greater than 20:4/18:2(n-6). The suppression of triacylglycerol synthesis by 20:5(n-3) was due to an increased (P less than 0.05) diacylglycerol specific activity, which indicates that 20:5(n-3) suppression of hepatic triacylglycerol production may be caused in part by the inhibition of diacylglycerol acyltransferase.     

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.     

Changing Fatty Acid Content of Growth Cone Lipids Prior to Synaptogenesis

The developing mouse was used to assess biochemical changes in membrane lipids during the period when nerve growth cones become synapses. Growth cone particles and synaptosomes were simultaneously obtained from common brain homogenates. Incorporation of the essential fatty acid, docosahexaenoic acid (22:6 omega-3), was correlated with the developmental changes in endogenous fatty acid content of growth cones and synaptosomes. Analysis of endogenous lipid content indicated that, at all ages studied, the growth cones contained more arachidonoyl acyl chains (20:4 omega-6) than did synaptosomes. Before the onset of synaptogenesis, levels of arachidonoyl chains increased and levels of 22:6, oleoyl and linoleoyl chains decreased in synaptosomes. Although stearoyl and palmitoyl (16:0) remained stable in synaptosomes, 16:0 decreased in growth cones. With the exception of 16:0 and 20:4, endogenous fatty acyl content of growth cones and synaptosomes became similar by postnatal day 10, which coincides with the onset of synaptogenesis. When 5-day-old mouse pups were injected intraperitoneally with [3H]22:6, the incorporation into growth cone and synaptosome phospholipids was greatest in phosphatidylethanolamine, followed by phosphatidylserine and phosphatidylcholine. Nominal labeling was present in phosphatidic acid and phosphatidylinositol. Labeling in neutral lipids was less than that of phospholipids, with triacylglycerol incorporating most of the neutral lipid label, followed by diacylglycerol and free 22:6. Only the growth cone fraction contained detectable amounts of 22:6-labeled cholesterol esters. The distribution of 22:6 label in plasma 72 h after injection indicated that approximately 60% of the label was in phospholipids with approximately 40% in neutral lipids and less than 5% in free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.     

Glutathione modulates lipid composition of human colon derived HT-29 cells

Glutathione (GSH) is important in maintaining intracellular thiol status. The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells. GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM). GSH was restored during early periods of cell growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC). Lipids were analysed following GSH depletion and supplementation. Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells. There were no detectable free fatty acids either in control or GSH-depleted cells. Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed. These changes were almost completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine. These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.     

Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.     

Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors.

A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.     

Analysis of lipids by gas-liquid chromatography and complementary methods in four strains of Aedes aegypti mosquitoes

Fatty acid composition of total lipids, neutral lipids and phospholipids of strains of Aedes aegypti were determined. The fatty acid composition of the strains differed quantitatively with regard to the relative percentage of commonly occurring fatty acids. Gas-liquid chromatography of fatty acid methyl esters showed 18:1 (oleic or elaidic) to be the predominant fatty acid. The fatty acid was identified as oleic by argentation thin-layer chromatography. A modified colorimetric method was used to determine tissue-free fatty acids. The lipids were predominantly triacylglycerol with lesser amounts of free fatty acids and decreasing amount of sterol ester, sterol, monoacylglycerol, diacylglycerol and hydrocarbons. The data show considerable lipid differences between the Caribbean strains (Les Cayes, Haiti, and San Juan, Puerto Rico) and the Jakarta (Indonesia) strain. The Shimba Hills (Kenya) strain was more similar to Jakarta than to the Caribbean strains. The results obtained with the different strains are discussed in relation to the established oral susceptibility to Dengue 1 and Dengue 2, yellow fever, and genetic analysis by isoenzyme studies.     

Alteration of lipid organization following fertilization of sea urchin eggs

The fluorescent probe merocyanine 540 was used to examine the organization of the lipids in the external leaflet of the plasma membrane after fertilization of sea urchin eggs. These lipids in unfertilized eggs are closely packed, as evidenced by their inability to bind the dye, whereas in fertilized eggs and cells of embryos up to at least the gastrula stage, the membrane becomes more loosely organized, and stains with bright ring fluorescence. Induction of late fertilization events with ammonia failed to induce this change in staining behavior. Sperm components are not required to induce this alteration since parthenogenetically activated eggs stained. However, treatment of eggs with procaine, which specifically inhibits the early event of cortical granule fusion, was effective in suppressing staining. These results indicate that cortical granule fusion after fertilization results in a change in the organization of the lipids of the plasma membrane of sea urchin eggs.     

Incorporation of [1-14C]Palmitic Acid into Neutral Lipids and Phospholipids of Rat Cerebral Cortex In Vitro

Abstract: Incorporation of [1-¹⁴C]palmitic acid into neutral lipids and phospholipids of rat cerebral cortex was examined in vitro in normal Krebs-Ringer bicarbonate buffer containing 3% (wthol) albumin and 0.75 mM palmitic acid. Under standard assay conditions, radioactivity in the triacylglycerol fraction increased rapidly during the first 30 min, and then decreased after 60 min, with corresponding increase in radioactivity in phosphatidyl choline, phosphatidyl ethanolamine, and a fraction of phosphatidyl inositol plus phosphatidyl serine. Diacylglycerol was shown to be an intermediate metabolite. Radioactivity increased in triacylglycerol, and decreased in phosphatidyl choline and phosphatidyl ethanolamine throughout incubation under NZ gas. In the fraction of phosphatidyl inositol plus phosphatidyl serine, radioactivity decreased after 30 min during incubation under N, gas. A possible acylation-deacylation cycle, in which triacylglycerol could be a source of free fatty acids for phospholipids, is discussed.     

Reversibility of Propranolol-Induced Changes in the Biosynthesis of Monoacylglycerol, Diacylglycerol, Triacylglycerol, and Phospholipids in the Retina

Abstract: The biosynthesis and metabolism of phospholipids and neutral glycerides were studied in the bovine retina. Radioactive glycerol was used as a precursor. Phentolamine and d - and dl -propranolol were found to produce similar effects on lipid metabolism in the retina. Marked stimulation of phosphatidylinositol (PhI) synthesis and maximal inhibition of phosphatidylcholine (PhC), diacylglycerol (DG), and triacylglycerol (TG) formation were observed within 5 min after exposure to 0.5 m M dl -propranolol. Pulse-chase experiments showed a high turnover rate in DG and a reversibility of the propranolol-induced changes produced during the synthesis of PhC, TG, DG, monoacylglycerol (MG), and phosphatidylserine. All reversals of the drug-induced biosynthetic profiles approached control values 60 min after incubation in drug-free medium. However, complete reversal was not achieved in any of the cases under these conditions. Propranolol appeared to inhibit both the formation of DG from phosphatidic acid and the further metabolism of DG, probably to MG. Phosphatidylethanolamine biosynthesis showed some recovery from this inhibition. Synthesis of Phi was greatly stimulated by preincubation with propranolol and was further enhanced by reincubation in the presence of propranolol. However, this effect was not reversed by reincubation without the drug. The active de novo biosynthesis of retinal phospholipids and glycerides is a very dynamic pathway that may be redirected by amphiphilic drugs. In addition, the partial reversal of modifications induced in the flux of [2-³H]glycerol through the lipids can occur during short-term reincubations of retinas in drug-free medium.     

Regulation of FFA by the acyltransferase pathway in focal cerebral ischemia-reperfusion

Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the ischemia period and at later time periods after reperfusion. In this study, the focal cerebral ischemia model was used to examine incorporation of [¹⁴C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. The label was injected intracerebrally into left and right MCA cortex 1 hr prior to decapitation. Labeled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 hr) after a 60 min ischemia, an inhibition of labeled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. When compared to labeled PC, there was a 3–4 fold increase in incorporation of label into phosphatidic acid and triacylglycerols (TG) in the right MCA cortex, suggesting of an increase in de novo biosynthesis of TG. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 hr after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, due mainly to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A₂ activity with both syaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia-reperfusion.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - PEpl ethanolamine plasmalogen - PI phosphatidylinositol - PS phosphatidylserine - poly-PI polyphosphoinsoitide - DG diacylglycerol - TG triacylglycerol - FFA free fatty acids - PUFA polyunsaturated fatty acids - MCA middle cerebral artery - CCAs common carotid arteries - HPTLC high performance thin layer chromatography - GLC gas-liquid chromatography - PLA₂ phospholipase A₂ Special issue dedicated to Dr. Leon S. Wolfe.     

Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.