Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.     

Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.     

Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.     

Resistance to ethanol disordering of membranes from ethanol-fed rats is conferred by all phospholipid classes.

Phospholipids extracted from liver microsomes and mitochondria of ethanol-fed rats retained the resistance to membrane disordered by ethanol which is observed in the intact isolated membranes. The lipid extracts were separated into the major phospholipid classes (phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol from microsomes and phosphatidylcholine, phosphatidylethanolamine and cardiolipin from mitochondria) by preparative TLC. The extent of membrane disordering by ethanol of phospholipid vesicles composed of a mixture of phospholipids from ethanol-fed rats and controls was determined from the reduction of the order parameter of the spin-probe 12-doxyl-stearate. In contrast to previous reports, we found that all phospholipid classes from ethanol-fed rats confer resistance to disordering by ethanol. To a first approximation the extent of resistance was proportional to the fraction of lipids from ethanol-fed rats, regardless of the phospholipid head-group. Subtle differences between phospholipid classes may exist but were too small to measure accurately. Except for phosphatidylethanol, incorporation of anionic phospholipids did not have a significant effect on the sensitivity of phospholipid vesicles to the disordering effect of ethanol. Vesicles prepared from mixtures of various dioleoyl phospholipids and natural phospholipids did not indicate a clear effect of fatty acid saturation on the sensitivity to disordering by ethanol. Although the precise molecular changes that occur in phospholipids from ethanol-fed rats have not been fully characterized it appears that subtle changes in all phospholipid classes contribute to the resistance to ethanol disordering of these membranes.     

Studies of the biochemical basis of steroid sulphatase deficiency--III. Phospholipid composition of microsomes from normal and steroid sulphatase deficient placentas

The phospholipid composition has been determined for placental microsomes from 11 normal and eight pregnancies complicated by steroid sulphatase deficiency. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin were found to be the major phospholipids of normal placental microsomes, comprising respectively 41.6 +/- 4.6% (mean +/- SD). 30 +/- 5.7% and 22.5 +/- 4.9% of the total phospholipid content. There was no correlation between the steroid sulphatase activity of the microsomes and the content of any of the three phospholipids. Though their contents were significantly decreased. (P less than 0.001) phosphatidylcholine, phosphatidylethanolamine and sphingomyelin similarly constituted the major portion of the total phospholipids in sulphatase deficient microsomes, representing 36 +/- 4.2%, 34 +/- 6.1% and 22.4 +/- 6.7% respectively. Only the percentage of phosphatidylcholine was significantly different (P less than 0.02) from normal microsomes. The results show that the decreased phospholipid content of steroid sulphatase deficient placental microsomes reflects a lower content of all major classes of phospholipids, particularly phosphatidylcholine.     

Intracellular phospholipid movement and the role of phospholipid transfer proteins in animal cells

The mechanism of the intracellular movement of phospholipids from their site of synthesis in the endoplasmic reticulum to mitochondria and other cell membranes is a major unsolved problem of cell biology. Phospholipid transfer proteins of varying specificity found in the soluble supernatant fractions of many tissues catalyze the transfer of phospholipids from microsomes to mitochondria in vitro. They are postulated to play a similar role in vivo, but evidence for their function in living cells is lacking. We have now used an analogue of choline, N-propyl-N,N-dimethylethanolamine [PDME, (2-hydroxyethyl)dimethylpropylammonium hydroxide], to devise a test for the function of the transfer proteins in living cells. The rates of translocation of newly synthesized phosphatidylcholine and the analogue phosphatidyl-PDME in living cells were compared with the rates of transfer in vitro catalyzed by soluble transfer proteins extracted from the same cells. Labeled PDME, choline, and ethanolamine were found to be rapidly incorporated into the lipids of isolated rat hepatocytes and of baby hamster kidney (BHK-21) cells in culture. The translocation of newly synthesized phosphatidylcholine and phosphatidyl-PDME was very rapid in both types of cells with a half-time for equilibration of a few minutes, while the translocation of phosphatidylethanolamine was much slower, with a half-time 20-80 fold longer than those of the other two phospholipids. We then compared these relative rates of movement with the activities of the phospholipid transfer proteins of the respective cells. Partially purified phosphatidylcholine transfer protein from rat liver transfers phosphatidylcholine and phosphatidyl-PDME at identical rates but transfers phosphatidylethanolamine at a rate too low to be detected. This result is consistent with an essential function of this transfer protein in vivo. In contrast, partially purified phosphatidylcholine phospholipid transfer protein from BHK cells transfers phosphatidylcholine rapidly, while no transfer of phosphatidyl-PDME and phosphatidylethanolamine was detected. We further found that the specific phosphatidylcholine transfer protein of BHK cells accounts for nearly all of the transfer activity detected in the crude soluble fraction. The rapid translocation of phosphatidyl-PDME in vivo in BHK cells is therefore inconsistent with the postulate that soluble phospholipid transfer proteins are responsible for the rapid movement of phospholipids from microsomes to mitochondria in living cells.     

Effects of analogues of ethanolamine and choline on phospholipid metabolism in rat hepatocytes

1. Analogues of ethanolamine and choline were incubated with different labelled precursors of phospholipids and isolated hepatocytes and the effects on phospholipid synthesis were studied. 2. 2-Aminopropan-1-ol and 2-aminobutan-1-ol were the most efficient inhibitors of [(14)C]ethanolamine incorporation into phospholipids, whereas the incorporation of [(3)H]choline was inhibited most extensively by NN-diethylethanolamine and NN-dimethylethanolamine. 3. When the analogues were incubated with [(3)H]glycerol and hepatocytes, the appearance of (3)H in unnatural phospholipids indicated that they were incorporated, at least in part, via CDP-derivatives. The distribution of [(3)H]glycerol among molecular species of phospholipids containing 2-aminopropan-1-ol and 1-aminopropan-2-ol was the same as in phosphatidylethanolamine. In other phospholipid analogues the distribution of (3)H was more similar to that in phosphatidylcholine. 4. NN-Diethylethanolamine stimulated both the conversion of phosphatidylethanolamine into phosphatidylcholine and the incorporation of [Me-(14)C]methionine into phospholipids. Other N-alkyl- or NN-dialkyl-ethanolamines also stimulated [(14)C]methionine incorporation, but inhibited the conversion of phosphatidylethanolamine into phosphatidylcholine. This indicates that phosphatidyl-NN-diethylethanolamine is a poor methyl acceptor, in contrast with other N-alkylated phosphatidylethanolamines. 5. These results on the regulation of phospholipid metabolism in intact cells are discussed with respect to the possible control points. They also provide guidelines for future experiments on the manipulation of phospholipid polar-headgroup composition in primary cultures of hepatocytes.     

Negatively charged phospholipids and their position in the cholesterol affinity sequence

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyelin with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin.From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin ? phosphatidylserine, phosphatidylglycerol > phosphatidylcholine ? phosphatidylethanolamine.     

Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [³H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [³H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [³H]-benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.     

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.     

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.     

Subcellular incorporation of 32P into phosphoinositides and other phospholipids in isolated hepatocytes

Isolated rat hepatocytes were incubated with 32Pi for various times and then fractionated into plasma membranes, mitochondria, nuclei, lysosomes, and microsomes by differential centrifugation and Percoll density gradient centrifugation. The phospholipids were isolated and deacylated by mild alkaline treatment. The glycerophosphate esters were separated by anion exchange high pressure liquid chromatography and assayed for radioactivity. It was found that plasma membranes, mitochondria, nuclei, lysosomes, and microsomes displayed similar rates of 32P incorporation into the major phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. This suggests that the phospholipids of these organelles are undergoing rapid turnover and replacement with newly synthesized phospholipids from the endoplasmic reticulum. However, the plasma membrane fraction incorporated 32P into phosphatidylinositol 4-phosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) at rates 5-10 and 25-50 times, respectively, faster than any of the other subcellular fractions. Although the plasma membrane is the primary site of 32P incorporation into DPI and TPI, this study also demonstrates that significant incorporation of 32P into DPI occurs in other subcellular sites, especially lysosomes.     

Diabetes affects phospholipid content in the nuclei of the rat liver.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on the content of different phospholipids and the incorporation of blood-borne 14C-palmitic acid into the phospholipid moieties in the rat liver nuclei. Diabetes was produced by intravenous administration of streptozotocin, and determinations were carried out two and seven days thereafter. Phospholipids were extracted from isolated nuclei and separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. Following that, they were quantified and radioactivity was measured. It was found that, in comparison to non-diabetic controls, two-day diabetes reduced the total content of phospholipids in the nuclei by 9.6%. The content of phospholipids in the nuclei by 9.6%. The content of phosphatidylcholine and phosphatidylserine was reduced and the content of the remaining phospholipids was stable. The specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin, based on radioactivity incorporated from 14C-palmitic acid, was elevated. Seven-day diabetes resulted in a reduction of the total phospholipid content in the nuclei by 39.4%. This was accounted for by a reduction in the content of each phospholipid fraction with the exception of cardiolipin. The specific activity of each phospholipid fraction, was elevated in this group. It is concluded that insulin is involved in the regulation of the nuclear phospholipid content.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

Schistosoma mansoni: synthesis and release of phospholipids, lysophospholipids, and neutral lipids by schistosomula

Lipids in the two surface membranes of Schistosoma mansoni may play an important role in the parasite's defense against host immunity. In particular, lysophosphatidylcholine lyses erythrocytes attached to the parasite and alters the lateral mobilities of their membrane proteins and lipids (Golan et al. 1986). Here, we have studied the incorporation of radiolabeled precursors into the major lipid classes of schistosomula as well as into lipids released by schistosomula into the medium. Radiolabeled polar head groups (choline and ethanolamine) and fatty acid precursors (palmitate and oleate) were linearly incorporated into parasite phospholipids. Fatty acids were differentially incorporated into the various phospholipid classes, principally into phosphatidylcholine and, to a lesser extent, into phosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine. The major neutral lipid class labeled, triglycerides, had a decrease in specific activity with time after pulse labeling and the specific activity of the phospholipids increased with time. Thus, triglycerides may provide acyl chains for phospholipid synthesis. Choline was incorporated into phosphatidylcholine and lysophosphatidylcholine, and ethanolamine into phosphatidylethanolamine and lysophosphatidylethanolamine. No evidence was found for phospholipid methylation or demethylation in schistosomula. Labeled lipids were linearly and selectively released into the medium. Triglycerides were released at the highest rate with measurable quantities of phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine also observed. Monopalmitoylphosphatidylcholine was the only lysophosphatidylcholine present in the medium as demonstrated by reverse-phase chromatography of released choline-labeled lysophosphatidylcholine. These studies demonstrate that schistosomula synthesize phospholipids and neutral lipids and release some of them into the culture medium. In particular, they release a single molecular species of a potent biologically active molecule, monopalmitoylphosphatidylcholine, that may play a role in the parasite's evasion of the immune response.     

Phospholipid metabolism in the brown alga, Fucus serratus

The metabolism of phospholipids in the brown alga, Fucus serratus was studied. The major phospholipids of this alga are phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and phosphatidylcholine. When the time-course of labelling of the lipids from [³²P] orthophosphate was studied, total labelling was approximately linear for 8 hr. All the major classes of phospholipid were labelled. The extent and pattern of labelling were not affected by the presence of proteins synthesis inhibitors phosphatidic acid was highly labelled at short time intervals. Phosphatidylcholine was relatively poorly labelled. The extent and pattern of labelling were not affected by the presence of protein synthesis inhibitors indicating that the enzymes involved in phospholipid synthesis have a rather slow turnover. Incorporation of radioactivity into phosphatidylglycerol was stimulated significantly by light.     

Alterations of new methylated phospholipid synthesis in the plasma membranes of macrophages exposed to chemoattractants

Chemotactic factors have been shown to inhibit the methylation of phosphatidylethanolamine in macrophages without affecting total phospholipid synthesis. It would thus be anticipated that newly synthesized membranes of macrophages exposed to chemoattractants would have an increased ratio of phosphatidylethanolamine to its methylated derivatives. These ratios were measured directly in newly synthesized phospholipids of plasma membranes isolated from guinea pig peritoneal macrophages. The phosphatidylethanolamine: methylated phospholipid ratio in such plasma membranes was increased by 53 to 111% upon exposure of the cells to chemotactic factors. This increase was due to decreased synthesis of methylated phospholipids and not to altered formation of phosphatidylethanolamine or activation of phospholipases. Methylated phospholipid ratios were also studied in the leading front lamellipodia isolated from macrophages migrating under chemotactic and nonchemotactic conditions. The phosphatidylethanolamine:methylated phospholipid ratios were increased up to fourfold in lamellipodia of macrophages migrating towards chemotactic agents when compared to those from cells migrating randomly. Biophysical changes in the plasma membrane produced by an increase in the ratio of phosphatidylethanolamine:methylated phospholipids as a result of exposure of cells to chemoattractants may be required for sustained directed migration.     

Effect of acute thioacetamide administration on rat brain phospholipid metabolism

Brain phospholipid composition and the [³²P]orthophosphate incorporation into brain phospholipids of control and rats treated for 3 days with thioacetamide were studied. Brain phospholipid content, phosphatidylcholine, phosphatidylethanolamine, lysolecithin and phosphatidic acid did not show any significant change by the effect of thioacetamide. In contrast, thioacetamide induced a significant decrease in the levels of phosphatidylserine, sphingomyelin, phosphatidylinositol and diphosphatidylglycerol. After 75 minutes of intraperitoneal label injection, specific radioactivity of all the above phospholipids with the exception of phosphatidylethanolamine and phosphatidylcholine significantly increased. After 13 hours of isotope administration the specific radioactivity of almost all studied phospholipid classes was elevated, except for phosphatidic acid, the specific radioactivity of which did not change and for diphosphatidylglycerol which showed a decrease in specific radioactivity. These results suggest that under thioacetamide treatment brain phospholipids undergo metabolic transformations that may contribute to the hepatic encephalopathy induced by thioacetamide.     

The microtubule analog protein, FtsZ, in the endosymbiont of trypanosomatid protozoa

Blastocrithidia culicis and Crithidia deanei are trypanosomatids that harbor an endosymbiotic bacterium in their cytoplasm. In prokaryotes, numerous proteins are essential for cell division, such as FtsZ, which is encoded by filament-forming temperature-sensitive (fts) genes. FtsZ is the prokaryotic homolog of eukaryotic tubulin and is present in bacteria and archaea, and has also been identified in mitochondria and chloroplasts. FtsZ plays a key role in the initiation of cytokinesis. It self-assembles into the Z ring, which establishes the division plane during septation. In this study, immunoblotting analysis using a FtsZ polyclonal antibody, revealed a 40-kDa band characteristic of FtsZ in endosymbiont fractions and in whole trypanosomatid homogenates, but not in whole cell extracts of aposymbiotic strains. Confocal microscopy and ultrastructural analysis revealed a specific and dispersed labeling over the endosymbiont. Bars and ring-like structures, which are suggestive of the presence of Z-rings, were never observed, even during the division of the symbiont. This peculiar distribution of FtsZ may represent an arrangement of cytoskeleton protein intermediate between prokaryotic and eukaryotic cells. The endosymbiont ftsz gene was completely sequenced after amplification of DNA from symbiont-bearing trypanosomatids or from pure endosymbiont fractions, using PCR and specific primers. The sequences obtained from the endosymbionts from C. deanei and B. culicis were very similar, and were most closely related to bacteria from the genus Pseudomonas.