Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.     

Lipid composition of miniature pig platelets

1. Analyses of platelet lipid composition were carried out on material pooled from male and female miniature pigs. 2. The cholesterol/phospholipid molar ratio was 0.6. 3. Phosphatidylcholine represents the major class of phospholipids (47%) and phosphatidylinositol the minor (2%). 4. The main fatty acids of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin were: palmitic, stearic, oleic, linoleic and arachidonic acids. 5. The ratios of saturated to unsaturated fatty acids were: sphingomyelin, 1.7; phosphatidylcholine, 1.2; phosphatidylserine, 0.9; phosphatidylethanolamine and phosphatidylinositol, 0.6. 6. Our results suggests that human and miniature pig platelet lipids bear several characteristics in common. This fact would allow miniature pig to be used as a new experimental model.     

Characterization and comparison of lipids in different squid nervous tissues

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.     

The high turnover rate of phosphatidylinositol in chicken heart ventricular cells during the earlier stages of development

The number of the chicken ventricular cells develops exponentially up to Day 12 in the developmental stages of embryos, after which the number gradualy decreases. The phosphatidylinositol metabolism at various stages in development (Days 5 to 21) has been studied. Ventricular cells were incubated in a physiological solution containing ³²P_i, or [1,3-³H]glycerol. Radioactivities incorporated into the phosphatidylinositol were estimated. The specific activity of [1,3-³H]glycerol, taken into phosphatidylinositol, at Day 12, was shown to be approximately equal to that of the other classes of phospholipids. However, the rate of labeling of ³²P_i into the phosphatidylinositol was extremely high in comparison with the other classes of phospholipids in the same ventricles. These results show that there is a rapid turnover of the phosphorylinositol moiety in the phosphatidylinositol in the earlier stages of development. This high turnover rate of the phosphatidylinositol was observed up to Day 12, after which it began to decrease. This turning point of the phosphatidylinositol metabolism coincided well with the decrease of the rate of cell proliferation. Therefore, this rapid turnover of phosphatidylinositol could have a specific functional role related to cell division. This rapid turnover of the phosphorylinositol moiety of the phosphatidylinositol associated with ventricular cell proliferation at different embryonic stages is reported for the first time.     

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32Pi, the incorporation of 32Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [3H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U-14C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold whereas the turnover of newly synthesized phosphatidylserine was normal. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine. These results demonstrate that exogenous phosphatidylserine can be efficiently incorporated into Chinese hamster ovary cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.     

Transverse asymmetry of phospholipids in subcellular membranes of rat liver

Subcellular membranes isolated from rat liver in a form impermeable to macromolecules were treated with phospholipase A₂ from Naga naja venom. The phosphatidylserine, phosphatidylethanolamine and about half of the phosphatidylcholine of microsomes, Golgi membranes, inner mitochondrial membranes, lysosomes and nuclear membranes were hydrolyzed. It is proposed that these phospholipids are localized in the outer surface of the membrane bilayer, which represents the cytoplasmic side in the living cell, while the remaining phosphatidylcholine and most of the phosphatidylinositol, sphingomyelin and cardiolipin may be assigned to the inner side of the bilayer.     

Phospholipids of liver cell nuclei during hibernation of Yakutian ground squirrel

In hibernating Yakutian ground squirrels S. undulatus, the content of total phospholipids in the nuclei of liver increased by 40% compared to that in animals in summer. In torpid state, the amount of sphingomyelin increased almost 8 times; phosphatidylserine, 7 times; and cardiolipin, 4 times. In active “winter” ground squirrels, the amount of sphingomyelin, phosphatidylserine, and cardiolipin decreased compared to the hibernating individuals but remained high compared to the “summer” ones. The torpor state did not affect the amount of lysophosphatidylcholine and phosphatidylinositol.     

Qualitative analysis of phospholipids isolated from nonviable plasmodium antigen

Plasmodium berghei infected mouse blood, and Plasmodium knowlesi infected monkey blood were processed by the French Press to prepare Antigen “A,” a parasitic fraction known to impart immunity, and Antigen “B,” a byproduct of Antigen “A” production. Normal mouse erythrocyte material was also prepared.The lipoidal material from these preparations was extracted using chloroform: methanol (1:1) and concentrated under nitrogen. This material was resuspended in chloroform and thin-layer chromatography was used to separate and identify the phospholipids therein. Antigen “A” contained sphingomyelin, phosphatidylcholine, phosphatydylinositol, and phosphatidylethanolamine. Antigen “B,” and uninfected mouse erythrocytic material, contained sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and possibly phosphatidylinositol. The absence of phosphatidylserine in Antigen “A” its presence in Antigen “B,” and in normal mouse material indicates that the protective Antigen “A” is free of host erythrocytic membrane fragments.     

Lipid composition of Trypanosoma cruzi.

1. Lipid composition of Trypanosoma cruzi epimastigote form in culture consist of 35% of phospholipids and 65% of neutral lipids. 2. Among the phospholipids, phosphatidylcholine is the more abundant (44%), followed by phosphatidylethanolamine (28%), phosphatidylinositol (12%), sphingomyelin (4%), and smaller amounts of cardiolipin, phosphatidic acid, lysolecithin, phosphatidylserine (traces), and an unidentified phospholipid (3%). 3. Pulse labeling with 32P showed highest specific incorporation in phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylcholine, suggesting a more active role for phosphatidylethanolamine in these organisms.     

Aminophospholipid translocase of human erythrocytes: phospholipid substrate specificity and effect of cholesterol

The outside-inside translocation rate and transmembrane equilibrium distribution, at 37 degrees C, of 16 different amphiphilic spin-labeled phospholipids have been determined in human erythrocytes. The transmembrane distribution was assessed by bovine serum albumin extraction of the spin-labels present in the outer monolayer. Within 15 min, more than 90% of the phosphatidylserine analogue was found in the inner monolayer; the equilibrium distribution of phosphatidylethanolamine spin-label was approximately 85-90% inside, with a half-time for translocation of approximately 50 min. In contrast, phosphatidylcholine reached a distribution corresponding to approximately 30% of the labels inside with a half-time of approximately 8 h, and only traces of sphingomyelin were found in the inner monolayer after 16 h. Thus, the spin-label analogues distributed themselves like endogenous phospholipids in red cells with a spontaneous segregation between the amino lipids and the choline-containing phospholipids. Progressive methylation of the amine group of phosphatidylethanolamine resulted in a stepwise decrease of the specific transport; modification of the beta-carbon of the serine also decreased the efficiency of the rapid translocation without abolishing it. Phosphatidyl-propanolamine was not transported. Substitution of the glyceride group by a ceramide abolished the rapid outside-inside translocation even with a molecule bearing a serine head group. Also it was found that esterification of the sn-2 position of the glycerol component was necessary for a rapid translocation since lysophosphatidylserine was only slowly transported from outside to inside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane lipids composition and metabolism during early embryonic development. Phospholipid subcellular distribution and 32P labeling

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented about 7% of the total phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gastrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk platelet fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.     

Phospholipid asymmetry during erythropoiesis. A study on Friend erythroleukemic cells and mouse reticulocytes

The distribution of phospholipids over the outer and inner layers of the plasma membranes of differentiated Friend erythroleukemic cells (Friend cells) and mouse reticulocytes has been determined. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol were found to be distributed symmetrically over both layers, sphingomyelin was found to be enriched in the outer layer (80-85%) and phosphatidylserine appeared to be present mainly in the inner layer (80-90%) of the plasma membranes of differentiated Friend cells. The outer layer of reticulocyte membranes contains 50-60% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 82-85% of the sphingomyelin and 40-42% of the phosphatidylinositol. All of the phosphatidylserine is present in the inner layer. The results show, that the asymmetric distribution of phospholipids, typical for erythrocyte membranes, is partially apparent already at an early stage of erythropoiesis, the proerythroblast, while the final organization of phospholipid distribution takes place at some stage during enucleation of the enormoblast and release of the reticulocyte into the blood stream.     

Role of phospholipids in early ontogenesis of Arctic-Boreal species <Emphasis Type="Italic">Leptoclinus maculatus</Emphasis> (Stichaeidae)

The content of total phospholipids and their classes (phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin) of muscles (flesh) and lipid sac of different developmental stages of young fish the daubed shanny, Leptoclinus maculatus from Kongsfjord (Svalbard, Norway) in winter was studied. The content of phospholipids in flesh decreases with age on account of phosphatidylcholine and phosphatidylethanolamine that probably related to their role in morphogenesis during differentiation of tissues and organs. The content of phospholipids is lower than reserve lipids in the lipid sac. The level of phospholipids in the lipid sac compared to flesh increases with age of fish reaching the maximum in benthic juveniles. Variations of minor phospholipids content of young fish of the daubed shanny indicate their participation in biochemical mechanisms of adaptation realizing in specific and varying Arctic conditions.     

Blood Plasma Phospholipids and Cholesterol during Hibernation of the Long-Tailed Ground Squirrel

Seasonal changes in the levels of phospholipids, diglycerides, cholesterol, and total protein in the blood plasma were investigated during hibernation of the long-tailed ground squirrel Spermophilus undulatus. During the winter period, the levels of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin phospholipids (per 1 mg of plasma protein) were increased in both torpid and active ground squirrels by 70–80, 50, 600–700, 70, and 150–200%, respectively; the level of phosphatidylserine did not change in comparison to the summer period. The plasma phospholipid composition differed between hibernating and active summer animals: in winter, the phosphatidylcholine mol % decreased by 20%, phosphatidylinositol and phosphatidylethanolamine increased by 3–4 times, and the phosphatidylserine mol % decreased by 50%, while sphingomyelin and lysophosphatidylcholine did not change in comparison to summer animals. In hibernating ground squirrels, the plasma cholesterol levels increased by two times, the diglyceride content diminished by 60%, and the level of protein (in milligrams per 1 mL plasma) increased by 20%. The simultaneous increase in the levels of cholesterol and total phospholipids, as well as the pronounced specific changes in the levels of individual phospholipids in the blood plasma of hibernating ground squirrels, indicate the involvement of plasma lipoprotein lipids in the molecular mechanisms of adaptation to natural hypobiosis in mammals and a possible role of these mechanisms in systemic reactions to damaging factors.     

Effect of triiodothyronine on the content of phospholipids in the rat liver nuclei.

The aim of the present study was to examine the effect of triiodothyronine (T3) on the content of phospholipids and on the incorporation of blood-borne palmitic acid into the phospholipid moieties in the nuclei of the rat liver. T3 was administered daily for 7 days, 10 microg x 100 g(-1). The control rats were treated with saline. Each rat received 14C-palmitic acid, intravenously suspended in serum. 30 min after administration of the label, samples of the liver were taken. The nuclei were isolated in sucrose gradient. Phospholipids were extracted from the nuclei fraction and from the liver homogenate. They were separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. The content and radioactivity of each fraction was measured. It was found that treatment with T3 reduced the content of phosphatidylinositol and increased the content of cardiolipin in the nuclear fraction. In the liver homogenate, the content of phosphatidylinositol decreased and the content of phosphatidylethanolamine and cardiolipin increased after treatment with T3. The total content of phospholipids after treatment with T3 remained unchanged, both in the nuclear fraction and in the liver homogenate. T3 reduced the specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin and had no effect on the specific activity of sphingomyelin and phosphatidylinositol both in the fraction of the nuclei and the liver homogenate. It is concluded that excess of triiodothyronine affects the content of phospholipids in the nuclei. The changes in the content of phospholipids in the nuclei largely reflect changes in their content in the liver.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Sphingolipids and the formation of sterol-enriched ordered membrane domains

This review is focused on the formation of lateral domains in model bilayer membranes, with an emphasis on sphingolipids and their interaction with cholesterol. Sphingolipids in general show a preference for partitioning into ordered domains. One of the roles of cholesterol is apparently to modulate the fluidity of the sphingolipid domains and also to help segregate the domains for functional purposes. Cholesterol shows a preference for sphingomyelin over phosphatidylcholine with corresponding acyl chains. The interaction of cholesterol with different sphingolipids is largely dependent on the molecular properties of the particular sphingolipid in question. Small head group size clearly has a destabilizing effect on sphingolipid/cholesterol interaction, as exemplified by studies with ceramide and ceramide phosphoethanolamine. Ceramides actually displace sterol from ordered domains formed with saturated phosphatidylcholine or sphingomyelin. The N-linked acyl chain is known to be an important stabilizer of the sphingolipid/cholesterol interaction. However, N-acyl phosphatidylethanolamines failed to interact favorably with cholesterol and to form cholesterol-enriched lateral domains in bilayer membranes. Glycosphingolipids also form ordered domains in membranes but do not show a strong preference for interacting with cholesterol. It is clear from the studies reviewed here that small changes in the structure of sphingolipids alter their partitioning between lateral domains substantially.     

Membrane phospholipid asymmetry in Semliki Forest virus grown in BHK cells

The distribution of phospholipids across the membrane bilayer of Semliki Forest virus grown in BHK cells has been examined by treating the virus with bee venom phospholipase A₂ and sphingomyelinase C from Staphylococcus aureus. From the amounts of different phospholipids which are degraded rapidly (half-time about 1 min for phospholipase A₂) we calculate that in virus isolated 16 h after infection about 95% of sphingomyelin, 55% of phosphatidylcholine, 20% of phosphatidylethanolamine and less then 5% of phosphatidylserine is present on the outer leaflet of the virus envelope. Less than 5% of the virus was permeable to macromolecules before or after treatment with phospholipases as judged by accessibility of the genome to external ribonuclease. A much slower (half-time about 1 h) breakdown by phospholipase A₂ of originally inaccessible phosphatidylcholine and phosphatidylethanolamine appeared to be due to an enzyme-induced loss of lipid asymmetry since the original asymmetric distribution of phospholipids was maintained for several hours when the virus alone was incubated at 37°C. However, virus incubated for 20 h at 37°C showed a marked loss of phosphatidylethanolamine and phosphatidylserine asymmetry and a greater susceptibility to lysis by longer treatment with phospholipase A₂.     

On the disposition of phospholipids in freshly isolated myelin sheath preparations from bovine brain

The extent of availability of the four major classes of phospholipids in freshly isolated bovine brain myelin has been studied using phospholipase C (Clostridium perfringens), phospholipase A (bee venom), sphingomyelase (Staphalococcus aureus) and trinitrobenzenesulphonate. The results have been compared with those of a previous study (Gwarsha et al., 1984). Taken together, the data suggest that 47–67% of the phosphatidylcholine, 45–66% of the ethanolamine-containing phosphoglycerides, 52–55% of the spingomyelin and 10–32% of the phosphatidylserine + phosphatidylinositol in the fresh myelin is available to these probes and hence may be located at the external surface of the membrane bilayer.     

Effect of Long-Term Feeding with Acetyl-L-Carnitine on the Age-Related Changes in Rat Brain Lipid Composition: A Study by 31P NMR Spectroscopy

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.