Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.     

Microbial Assimilation of Hydrocarbons: Identification of Phospholipids

The distribution of phospholipids derived from Micrococcus cerificans was determined under a variety of nutritive conditions. Cells were grown with hexadecane, heptadecane, or acetate serving as the sole carbon source. Total lipid was isolated by chloroform-methanol extraction, and the phospholipid fraction was isolated by silicic acid column chromatography. The phospholipids were characterized by silicic acid chromatography, by thin-layer chromatography, and by identification of water-soluble products resulting from acid hydrolysis of purified phospholipids. Major phospholipids characterized were phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Minor phospholipids were phosphatidylglycerol phosphate and phosphatidylserine. Trace amounts of methylated derivatives of phosphatidylethanolamine were determined by incorporation of ¹⁴C from ¹⁴C-methylmethionine. These experiments demonstrated the presence of phosphatidyl-N-methylethanolamine, phosphatidyl-N,N′-dimethylethanolamine, and phosphatidylcholine in trace quantities. Pulse labeling with ¹⁴C-serine demonstrated the direct incorporation of serine into phosphatidylserine followed by decarboxylation to phosphatidylethanolamine.     

Studies of glucose metabolism in Hymenolepis diminuta using 13C nuclear magnetic resonance

The metabolism in vitro of U-¹³C-glucose and NaH¹³CO₃ by two strains of adult Hymenolepis diminuta, the ANU and UT strains, was examined using ¹³C n.m.r. spectroscopy. The incubation medium and perchlorate extracts from worms incubated in vitro with U-¹³C-glucose showed incorporation of significant quantities of label into the end products succinate, lactate and acetate, and also into glycogen. Similar experiments with NaH¹³CO₃ showed incorporation principally into succinate C-1,4, plus significant labelling also in lactate C-1. This shows that nutochondrial malate or pyruvate contributes to the cytosolic pyruvate pool in H. diminuta. The metabolism of U-¹³C-glucose was followed directly by incubating live worms directly in the spectrometer. Worms from 24 h-fasted hosts metabolised the added glucose completely during an experimental period of 2 h and incorporation of label was evident in the time course spectra. Parasites from fed hosts metabolised the added glucose more slowly. This work confirms the accepted routes of glucose metabolism in H. diminuta and demonstrates the utility of the n.m.r. technique in investigating the metabolism of parasites.     

Generation of formic acid and ethanolamine from serine in biosynthesis of linear gramicidin by a cell-free preparation of Bacillusbrevis (ATCC 8185)

A growing organism that produces antibiotic peptide was incubated with L-(U-¹⁴C)serine for labeling linear gramicidin. Linear gramicidin was isolated by a simple chromatographic method from tyrothricin (mixture of linear gramicidin and tyrocidine) applied to a column of basic aluminum oxide. The hydrolysate of labeled linear gramicidin on thin layer chromatography showed that L-(U-¹⁴C)serine was one of a precursor of ethanolamine moiety by autoradiography. L-(3-¹⁴C)serine generated formic acid in the presence of tetrahydrofolic acid by an enzyme fraction prepared with ammonium sulfate, and further formed ethanolamine binding to the protein. Formylvaline was biosynthesized by it with tetrahydrofolic acid and ATP, and subsequently released from the protein.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism

In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP-ethanolamine and its transfer to diglyceride, or by base-exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base-exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L-[G-3H]serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L-[U-14C]serine and [1-3H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole-brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.     

The role of glucose in the lipid metabolism of the rat tapeworm Hymenolepis diminuta

The composition of the neutral lipids and the phospholipids, and the role of glucose in the lipid metabolism of prepatent (12-day-old) Hymenolepis diminuta has been studied in vitro. Triglyceride was the most abundant lipid present; substantial amounts of sterol and sterol ester, diglyceride, free fatty acids and monoglycerides were also present. The phospholipids, which were qualitatively and quantitatively similar to those of other invertebrates and vertebrates, were, in order of abundance, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphoinositide, lysophosphatidylcholine, cardiclipin, phosphatidic acid, lysophosphatidic acid and phosphatidylglycerol. Small amounts of glucose carbon were incorporated into the lipids, principally the water soluble (glycerol) moiety of the triglycerides; only traces were incorporated into the phospholipids. Small amounts of glucose were converted to inositol and galactose. The principal pathway of triglyceride synthesis is suggested to be via the α-glycerophosphate-phosphatidic acid-diglyceride pathway.     

Synthetic pathways of glycerophospholipids in adult Brugia pahangi and Brugia patei

The pathways of glycerophospholipid syntheses in adult Brugia pahangi and Brugia patei were examined by radioisotopic incorporation and demonstration of the enzymatic steps. Radiolabelling studies showed that l-U-¹⁴C-glycerol-3-phosphate was rapidly incorporated into glycerophospholipids of B. pahangi and B. patei, respectively, with the label distributed in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and cardiolipin (CL) fractions. Crude extracts of these worms were found to contain significant activities of sn-glycerol-3-phosphate acyl-transferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), choline phosphotransferase (EC 2.7.8.2), ethanolamine phosphotransferase (EC 2.7.8.1), PE methyltransferase (EC 2.1.1.17), PS decarboxylase (EC 4.1.1.65), phosphatidylglycerolphosphate synthetase (EC 2.7.8.5), phosphatidylinositol synthetase (EC 2.7.8.11), and base exchange enzymes of ethanolamine, serine and inositol. These findings suggest that filarial worms can synthesize PC by two pathways, PE by three pathways, and PI by two pathways and fabricate PS, PG and CL.     

Base-exchange reactions for the synthesis of phospholipids in nervous tissue: the incorporation of serine and ethanolamine into the phospholipids of isolated brain microsomes

Abstract— The calcium-dependent incorporation of l -[3-³H]serine and [1,2-¹⁴C]ethanol-amine into the phospholipid of isolated subcellular fractions from chick brain was studied and the properties of incorporation were examined. The microsomal fraction was found to possess the highest rate of incorporation and was able to convert under optimal conditions about 120 nmol of labelled serine and 220 nmol of ethanolamine/g of fresh brain microsomes/h. The requirement for Ca²⁺ ion appeared to be absolute. Mg²⁺ ion caused a gradual reduction in the existing enzymic activity, only when pre-incubated with microsomes and labelled bases before adding Ca²⁺ ion. The incorporation of serine and ethanolamine was actively inhibited by Hg²⁺, Co²⁺, Cu²⁺ and Mn²⁺ ions, and was abolished by ethylenediamine tetra-acetate treatment. Ethanolamine, but not choline, inositol or carnitine, competitively inhibited serine incorporation, while d -serine had slight effect. Conversely, l -serine inhibited competitively the incorporation of ethanolamine. The greater part of the incorporated serine (85 per cent) was localized in microsomal phosphatidylserine, while a small percentage was found in phosphatidylethanolamine. Similarly, 90 per cent of the incorporated ethanolamine was confined to phosphatidylethanolamine and a small percentage was found in the plasmalogen derivative. The mechanism of serine and ethanolamine incorporation was investigated. The results are compared with those published for similar mammalian and non-mammalian systems.     

Phosphatidylethanolamine synthesis in adult Dirofilaria immitis females

Srivastava Arvind K., Jaffe Julian J. and Lambert Roger A. 1985. Phosphatidylethanolamine synthesis in adult Dirofilaria immitis females. International Journal for Parasitology15: 429–433. Adult Dirofiliaria immitis females were found able to synthesize phosphatidylethanolamine (PE) by way of the following three pathways: (1) phosphorylethanolamine, cytidine diphosphoethanolamine and 1,2-diacylglycerol; (2) decarboxylation of phosphatidylserine (PS); and (3) direct exchange of ethanolamine for choline or serine in preformed phosphatidylcholine or PS. The latter two pathways were confined to the paniculate fraction of worm homogenates. Under stated assay conditions, the respective rates of PE formation by way of these pathways in the order given were around 250, 8500 and 2–3 pmol min^?1 mg^?1 protein.     

The source of antigen in an adult tapeworm

Hopkins C. A. and Barr I. F. 1982. The source of antigen in an adult tapeworm. International Journal for Parasitology12: 327–333. Although a primary infection of Hymenolepis diminuta is not rejected for 9–15 days by a mouse, it has been shown that a primary infection terminated chemically after only 3 days induces as good protection against challenge. This demonstrated that a scolex and 1–2 mm of neck tissue (all that is formed by day 3 post infection) are an adequate source of ‘protective’ antigen. Irradiated (350 Gy) cysticercoids which survive but show little growth immunize as effectively as normal cysticercoids which indicates actively growing neck tissue is not essential and hence the scolex alone is a sufficient source of ‘protective’ antigens. In the rat irradiated cysticercoids were found to establish, double their length over 3–6 days and then slowly shrink, but 14% of the worms were still present 49 days p.i. Although a primary infection of normal worms in a rat markedly depresses growth of a secondary infection administered 7 days after the chemical expulsion of the primary, irradiated scoleces induced no measurable protection. These results are discussed in relation to the source of antigen and the fundamental difference in the protective response of mice (an abnormal host) and rats (a normal host) to the tapeworm H. diminuta in the small intestine.     

Formation of ceramide phosphorylethanolamine from phosphatidylethanolamine in the rumen protozoon Entodinium caudatum (Short Communication)

From `pulse'-labelling experiments of Entodinium caudatum with [¹⁴C]ethanolamine and by incubating the organism with [³²P]phosphatidylethanolamine it is concluded that phosphatidylethanolamine can act as a direct precursor of the phosphorylethanolamine moiety of ceramide phosphorylethanolamine. The phosphorylethanolamine is probably never liberated in the free form but is transferred directly to a ceramide or ceramide-containing acceptor. The results are also in agreement with previous conclusions that phosphatidylethanolamine is the direct lipid precursor of N-(1-carboxyethyl)phosphatidylethanolamine.     

Differential Utilization of the Ethanolamine Moiety of Phosphatidylethanolamine Derived from Serine and Ethanolamine during NGF-Induced Neuritogenesis of PC12 Cells

Neurite elongation involves the expansion of the plasma membrane and phospholipid synthesis. We investigated membrane phosphatidylethanolamine (PE) biosynthesis in PC12 cells during neurite outgrowth induced by nerve growth factor (NGF). When PE was prelabeled with [³H]ethanolamine and the radioactivity was chased by incubation with 1 mM unlabeled ethanolamine, the radioactivity of [³H]PE steadily declined and [³H]ethanolamine was released into the medium in NGF-treated cells during neurite outgrowth; in the absence of unlabeled ethanolamine, the radioactivity of [³H]PE remained relatively constant for at least 24 hr. In undifferentiated cells but not in NGF-treated cells, [³H]phosphoethanolamine accumulated in significant amounts during pulse labeling, and was converted partly to PE but largely released into the medium irrespective of incubation with unlabeled ethanolamine. The decline in the radioactivity of [³H]PE and release of [³H]ethanolamine following incubation with unlabeled ethanolamine were also observed in undifferentiated cells. Thus, the ethanolamine moiety of PE derived from ethanolamine is actively recycled in both differentiated and undifferentiated cells. When PE was derived from [³H]serine through phosphatidylserine (PS) decarboxylation, the decrease in radioactivity of [³H]PE and release of [³H]ethanolamine into the medium following incubation with unlabeled ethanolamine were observed only in NGF-treated cells, but not in undifferentiated cells, indicating that the ethanolamine moiety of PE derived from PS is actively recycled only in the cells undergoing NGF-induced neuritogenesis. Thus, in PC12 cells, the ethanolamine moiety of PE derived from PS is regulated differently from that of PE derived from ethanolamine.     

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[³H(G)]serine and [¹⁴C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.     

Cryptic diversity in hymenolepidid tapeworms infecting humans

An adult hymenolepidid tapeworm was recovered from a 52-year-old Tibetan woman during a routine epidemiological survey for human taeniasis/cysticercosis in Sichuan, China. Phylogenetic analyses based on sequences of nuclear 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit 1 showed that the human isolate is distinct from Hymenolepis diminuta and Hymenolepis nana, the common parasites causing human hymenolepiasis. Proglottids of the human isolate were unfortunately unsuitable for morphological identification. However, the resultant phylogeny demonstrated the human isolate to be a sister species to Hymenolepis hibernia from Apodemus mice in Eurasia. The present data clearly indicate that hymenolepidid tapeworms causing human infections are not restricted to only H. diminuta and H. nana.     

Enzymes of adenosine metabolism in Hymenolepis diminuta (Cestoda)

Gaitanaki C. and Beis I. 1985. Enzymes of adenosine metabolism in Hymenolepis diminuta (Cestoda). International Journal for Parasitology15: 651–654. The activities of 5-nucleotidase (E.C. 3.1.3.5), adenosine deaminase (E.C. 3.5.4.4), adenosine kinase (E.C. 2.7.1.20), AMP deaminase (E.C. 3.5.4.6) and adenylate kinase (E.C. 2.7.4.3) were demonstrated in homogenates of Hymenolepis diminuta. The K_m values for adenosine and AMP of the above enzymes were determined. The importance of these enzymes in the maintenance of adenosine concentration on a steady state in H. diminuta is discussed     

Extracellular vesicles secreted by model tapeworm Hymenolepis diminuta: biogenesis,ultrastructure and protein composition

We provided the first known evidence of the presence and release of extracellular vesicles in adults of important model tapeworm Hymenolepis diminuta. Two different subtypes have been observed on the surface of the worm and among the secretory products confirmed by several microscopical methods. Proteomic analysis revealed the presence of parasite-specific proteins as well as those of the host in purified extracellular vesicles. Among the protein cargo, we identified potential drug targets, vaccine candidates and H. diminuta antigens. Finally, the protein composition further revealed proteins participating in the endosomal complex required for transport-dependent biogenesis pathway.     

Complement-mediated lysis in vitro of newly excysted tapeworms: Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana and Hymenolepis citelli

Bøgh H., Christensen J.P.B. and Andreassen J. 1986. Complement-mediated lysis in vitro of newly excysted tapeworms: Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana and Hymenolepis citelii. International Journal for Parasitology16: 157–161. Newly excysted worms of Hymenolepis diminuta were lysed in 50% normal serum from all 13 animal species tested, including man. Since H. diminuta was neither lysed in complement inactivated serum—by heat or adding EDTA, LPS or CVF—nor in C5-deficient mouse serum, it is concluded that the lysis was associated with the complement cascade. It is shown that H. diminuta can activate the complement system via both the classical and alternative pathway. Furthermore, it is indicated that the lysis is independent of serum antibodies. Hymenolepis nana and H. citelli were also lysed in all normal sera tested, eight and six respectively, while newly excysted worms of H. microstoma were lysed in normal sera from 10 mammals and birds, but not in sera from its hosts, the mouse, rat and golden hamster. This indicates that the complement system of these three species differs from that of the other species tested in such a way that H. microstoma is able to avoid lysis in these sera.     

Synthesis of Ethanolamine and Its Regulation in Lemna paucicostata

The metabolism of ethanolamine and its derivatives in Lemna paucicostata has been investigated, with emphasis on the path-way for synthesis of phosphoethanolamine, a precursor of phosphatidylcholine in higher plants. In experiments involving labeling of intact plants with radioactive serine, ambiguities of interpretation due to entry of radioactivity into methyl groups of methylated ethanolamine derivatives were mitigated by pregrowth of plants with methionine. Difficulties due to labeling of diacylglyceryl moieties of phospholipids were avoided by acid hydrolysis of crucial samples and determination of radioactivity in isolated serine or ethanolamine moieties. The results obtained from such experiments are most readily reconciled with the biosynthetic sequence: serine → ethanolamine → phosphoethanolamine → phosphatidylethanolamine. A possible alternative is: serine → phosphatidylserine → phosphatidylethanolamine → ethanolamine → phosphoethanolamine. Cell-free extracts of L. paucicostata were shown to produce CO₂ from the carbon originating as C-1 of serine at a rate sufficient to satisfy the demand for ethanolamine moieties. A number of experiments produced no support for a hypothetical role for phosphoserine in phosphoethanolamine formation. Uptake of exogenous ethanolamine commensurately down-regulates the synthesis of ethanolamine moieties (considered as a whole, and regardless of their state of derivatization at the time of their formation). In agreement with previous observations, uptake of exogenous choline down-regulates the methylation of phosphoethanolamine, without being accompanied by secondary accumulation of a marked excess of ethanolamine derivatives.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.