[Non-valvular cardiac devices endocarditis]. / L'endocardite sur matériel de stimulation intracardiaque.

The risk of infective endocarditis on pacemaker or ICD is not negligible and has increased in recent years. Several host-related, procedure-related, or device-related risk factors have been recognized. Owing to its potential severity, the possibility of infective endocarditis should be envisaged in patients with repeated pulmonary infections or documented bacteremia and transesophageal echocardiography should then be used. The most common germs causing pacemaker endocarditis are staphylococci. Treatment requires prolonged antibiotic therapy and retrieval of the pacemaker and leads.

Topological distribution of choline phospholipid fatty acids in trout intestinal brush-border membrane.

The transbilayer distribution of choline phospholipids in trout intestinal brush-border membrane has been investigated using phospholipase C (from Clostridium welchii). In the middle intestine, 84% of phosphatidylcholine (PC) and 60% of sphingomyelin (SP) are located in the outer membrane leaflet. In the posterior intestine, 89% of PC and 52% of SP are located in the outer membrane leaflet. The externally located PC molecular species are (n - 3) fatty acid-rich in both parts of the intestine. While the sphingomyelin molecular species containing 24:1(n - 9) are exclusively located in the outer leaflet in the middle intestine, those containing 14:0 are more abundant in the same leaflet but in the posterior intestine. This strongly asymmetric distribution of both choline phospholipids may have numerous consequences on the brush-border membrane characteristics.

Phospholipase activities of rat brain cytosol. Occurrence of phospholipase C activity with phosphatidylcholine.

21-day-old rat brain contains a soluble phospholipase C with the ability to hydrolyse phosphatidylcholine. This enzyme has an alkaline pH optima. The results of the DEAE-cellulose fractionation, the pH profile and the Ca2+-dependency suggest that the enzyme may be the same as that responsible for phosphatidylinositol hydrolysis. The activity of the phospholipase C is associated closely with a diacylglycerol lipase. The two enzyme activities could be separated by DEAE-cellulose fractionation, resulting in greater than 100% apparent recovery of the phospholipase C acivity. This phospholipase C activity is not the result of the back reaction of choline phosphotransferase.

Topological distribution of aminophospholipid fatty acids in trout intestinal brush-border membrane.

The transbilayer distribution of aminophospholipids in trout intestinal brush-border membrane has been investigated using trinitrobenzene sulfonic acid (TNBS). In the middle intestine, phosphatidylethanolamine (PE) is symmetrically distributed between the two leaflets while 68% of the phosphatidylserine (PS) are located in the inner membrane leaflet. In the posterior intestine, 64% of the PE and 69% of the PS are located in the inner membrane leaflet. When asymmetrically distributed, the inner species of PE and PS have a higher content of 22:6(n-3) than the outer ones. This asymmetric distribution of docosahexaenoic acid in trout intestinal brush-border membrane might be related to the rod-like shape of the microvillus membrane and to its metabolism to hydroxylated derivatives.

Partial purification of ethanolaminephosphotransferase from rat brain microsomes.

Rat brain ethanolaminephosphotransferase (CDPethanolamine : 1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) was solubilized by treating rat brain microsomes with buffered solutions containing octyl glucoside or Triton X-100. The solubilized enzyme was stable both at 4 degrees C and at -18 degrees C. A partial purification was obtained using an ion-exchange chromatographic procedure. The partially purified enzyme showed four major bands in SDS-polyacrylamide gel electrophoresis; its specific activity was increased by a factor of 37 compared to that of the membrane-bound enzyme. Glycerol and diacylglycerol were effective as stabilizers. Phosphatidylcholine, lysophosphatidylcholine and phosphatidylserine increased both the specific activity and the stability of the partially purified enzyme.

Reversibility of the reactions catalyzed by cholinephosphotransferase and ethanolaminephosphotransferase solubilized from rat-brain microsomes.

The incorporation of CMP into CDP-ethanolamine and CDP-choline, catalyzed by ethanolaminephosphotransferase (EC 2.7.8.1) and cholinephosphotransferase (EC 2.7.8.2), respectively, has been studied in solubilized preparations of rat-brain microsomes. Mn2+ ions were required for the maximal activity of both enzymes. The CMP concentration needed to reach the half-maximal reaction rate was 1.6 microM for both activities. The rate of incorporation of CMP into CDP-choline and CDP-ethanolamine was increased by increasing the concentration of phosphatidylcholine and phosphatidylethanolamine, respectively, in detergent-phospholipid micellar systems. The rate of the reaction at pH 6.5 was comparable with that measured at pH 8.5, whereas the rate of synthesis of phosphatidylcholine and phosphatidylethanolamine, catalyzed by the same enzymes, increased with pH. Ethanolaminephosphotransferase, which catalyzes the synthesis of phosphatidylethanolamine from CDP-ethanolamine and diacylglycerol, was co-eluted with the enzyme activity catalyzing the reverse reaction, when solubilized microsomes were submitted to anion exchange chromatography on DEAE Bio-Gel A. Cholinephosphotransferase was inactivated during the chromatographic procedure.

Effects of deoxycholate and phospholipase A2 on choline and ethanolamine phosphotransferases of chicken brain microsomes.

Ethanolamine phosphotransferase (EC 2.7.8.1) and choline phosphotransferase (EC 2.7.8.2) activities were assayed in fresh microsomes from adult chicken brains with either diacylglycerols or alkylacylglycerols. Pretreatment of microsomes with 1.25 mM sodium deoxycholate, a concentration less than the critical micelle concentration, produced a slight inhibition of choline phosphotransferase activity. A deoxycholate concentration (5.0 mM) greater than the critical micelle concentration (3.0 mM) decreased the choline phosphotransferase activity by more than 70% but had no effect on ethanolamine phosphotransferase activity. Inclusion of 1.25 mM deoxycholate in the assay medium decreased choline phosphotransferase activity 35% but increased ethanolamine phosphotransferase activity 50%. The deoxycholate appeared to inactive the choline phosphotransferase. Phospholipase A2 (Vipera russelli) treatments of microsomes removed phosphoglycerides and decreased both phosphotransferase activities to a similar extent. Decreased activities are probably due to disruption of the membrane structure. Choline and ethanolamine phosphotransferase activities are apparently in different enzymes which lack specificity for the type of diglyceride. Thus, the systematic names should include 1,2-diradyl-sn-glycerol instead of 1,2-diacyl-sn-glycerol.

Effect of exogenous gangliosides on the lipid composition of chick neurons in culture.

When exogenous gangliosides are added to the growth medium of neuronal cell cultures they are inserted into their plasma membranes and are afterwards metabolized in the cytoplasmic interior. The action of exogenous gangliosides brings important morphological and biochemical changes to neurons in culture. The present report shows that the treatment with exogenous gangliosides of a primary culture of chick neurons modified the distribution of fatty acids in phosphatidylinositol (PI), mainly that of arachidonic acid and the fatty acids of the (n - 3) series without affecting the other phospholipids. The composition of neutral lipids did not change but their content was increased up to 2-3-fold depending upon the concentration of gangliosides. The change of the growth medium from one containing fetal calf serum to a chemically defined one reduced dramatically the content of free fatty acids while the addition of gangliosides raised this content to normal levels. The increase in the amount of diacylglycerol (DG) confirmed the finding that gangliosides stimulate phosphoinositide degradation. Finally the fatty acid composition of DG suggests indirectly that this compound might be produced also by degradation of phosphatidylcholine and not only of PI.

An improved procedure for the purification of ethanolaminephosphotransferase. Reconstitution of the purified enzyme with lipids.

Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminephosphotransferase was strongly modified by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.

Specific stimulation of phospholipase activities with phosphatidylethanolamine in transformed cells.

Phospholipase A1, A2 and C activities with phosphatidylethanolamine were enhanced in C6 cells relative to primary astrocytic cultures. Enhancement was a function of cell density. Phospholipase activities with phosphatidylcholine were unchanged as a function of cell density, while phospholipase C activity with phosphatidylinositol was reduced. All acid phospholipase activities measured were low or essentially absent in the three transformed cell lines examined. These results suggest that arachidonate release upon confluency is mainly from phosphatidylethanolamine.

Effect of neuraminidase treatment on the topological distribution of phospholipids in chick brain microsomes.

The desialylation of chick brain microsomal membranes affects the transbilayer distribution of phospholipids. When intact microsomes were treated with neuraminidase, less phosphatidylcholine and sphingomyelin could be hydrolysed with phospholipase C under experimental conditions which allowed the hydrolysis of the phospholipids of the external leaflet only. In contrast, the accessibility of phosphatidylethanolamine and phosphatidylserine to the external probes (trinitrobenzene sulfonic acid or phospholipase C) was not affected. After neuraminidase treatment of a microsomal fraction, less phosphatidylcholine, newly synthesized through the cytidine pathway, could be hydrolysed by phospholipase C, whereas the reaction of newly synthesized phosphatidylethanolamine molecules with trinitrobenzene sulfonic acid was not affected. The results suggest that in biological membranes some choline phospholipid molecules may interact with the sialyl residue of sialocompounds. This interaction may contribute to the maintenance of phospholipid asymmetry in brain membranes.

Compartmentation of membrane phosphatidylethanolamine formed by base-exchange reaction in rat brain microsomes.

The compartmentation of membrane phosphatidylethanolamine (PE) formed by base-exchange reaction in rat brain microsomal vesicles has been investigated. After labelling membrane PE by base-exchange in vitro, microsomal vesicles were treated with trinitrobenzenesulfonic acid (TNBS). The amount of membrane PE reacting with TNBS depends on the duration and the temperature of the reaction as well as on the TNBS concentration. It was found that almost all of the labelled PE molecules, but only about 24% of membrane PE, were accessible to TNBS, under very mild reaction conditions. It is concluded that PE labelled by base-exchange is completely localized in the cytoplasmic leaflet of microsomal vesicles.

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid.

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium welchii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.

Ether lipid content of phosphoglycerides from the retina and brain of chicken and calf.

Phospholipid contents and compositions were determined for chicken and calf retinas, chicken brain and calf gray matter. Retinal phospholipid compositions differ from brain phospholipid compositions by including a higher percentage of choline phosphoglycerides and lower percentages of ethanolamine and serine phosphoglycerides. The proportion of sphingomyelin is lower in calf retina than in calf brain. Among the ethanolamine phosphoglycerides, the proportions of the alk-1-enylacyl (plasmalogen) type are lower and the proportions of alkylacyl and diacyl types are higher in retina than in brain. The alkyacyl glycerophosphoethanolamines accounted for 7.6% and 8.9% of the ethanolamine phosphoglycerides from chicken and calf retinas respectively. Lower proportions of plasmalogens in the choline phosphoglycerides were found in retina as compared with brain. The alkylacyl glycerylphosphocholines comprised 4.0% of the retinal choline phosphoglycerides. Overall, a smaller proportion of retinal phosphilipids than of brain phospholipids contained alkyl or alk-1-enyl ether groups and the ratio of alkyl groups to alk-1-enyl groups was greater in retina than in brain.

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.

In vitro and in vivo ethanolamine metabolism in rat brain: effect of time and aging.

The effect of the time in culture of foetal rat neurons and age on the incorporation of radioactive ethanolamine into methylated derivatives was investigated. Decreased incorporation of [3H]ethanolamine into its various methylated water-soluble and lipidic derivatives was observed in rat neurons cultures at 12 day in vitro (DIV) as compared to the 3rd and the 7th DIV. In vivo studies showed that there was a diminished labeling of methylated products in the older animals as compared to the younger ones. These in vitro and in vivo observations suggest a generalized decrease of N-methyltransferase activities during maturation and aging.

Effect of D609 on phosphatidylcholine metabolism in the nuclei of LA-N-1 neuroblastoma cells: a key role for diacylglycerol.

In our previous studies, TPA treatment of LA-N-1 cells stimulated the production of diacylglycerol in nuclei, probably through the activation of a phospholipase C. Stimulation of the synthesis of nuclear phosphatidylcholine by the activation of CTP:phosphocholine cytidylyltransferase was also observed. The present data show that both effects were inhibited by the pretreatment of the cells with D609, a selective phosphatidylcholine-phospholipase C inhibitor, indicating that the diacylglycerol produced through the hydrolysis of phosphatidylcholine in the nuclei is reutilized for the synthesis of nuclear phosphatidylcholine and is required for the activation of CTP:phosphocholine cytidylyltransferase.

Acidic phospholipids inhibit the phospholipase D activity of rat brain neuronal nuclei.

An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC50 values varied from 3.5 microM for PIP2 to 200 microM for phosphatidic acid. The hydrolysis of PIP2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity.

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.

Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells.

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.