The polar lipid composition of Mesorhizobium ciceri

The extractable lipid composition of Mesorhizobium ciceri strain HAMBI 1750 grown in a phosphate sufficient medium (79CA) is reported. Cardiolipin (CL—27% of total lipids), phosphatidylglycerol (PG—18%), phosphatidylethanolamine (PE—1%), phosphatidylcholine (PC—30%) and two methylated derivatives of PE, i.e. phosphatidyl-N, N-dimethylethanolamine (DMPE—1%) and phosphatidyl-N-monomethylethanolamine (MMPE—1%), were found to make up the phospholipids of the analysed bacteria. Nonphosphorus, ornithine-containing lipid (OL—10%) was also detected. Polar groups of phospholipids were predominantly acylated with cis-11,12-methyleneoctadecanoyl (lactobacillic) residues, whereas the ornithine lipid contained mainly 3-hexadecanoyloxy-11,12-methyleneoctadecanoic acid bound to the α-amino group.     

The geometry of the thioester group and its implications for the chemistry of acyl coenzyme A

L-929 cell surface membranes were incubated with S-adenosyl-l-[methyl-³H]-methionine and found to contain phosphatidylethanolamine: S-adenosylmethionine N-methyltransferase (phosphatidylethanolamine N-methyltransferase) activity. The enzyme or combination of enzymes responsible for this activity methylated endogenous phosphatidylethanolamine and its methylated derivatives to yield phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Maximum enzyme activity was expressed at pH 6.9, the reaction was not dependent on the presence of divalent cations, and exogenously added phospholipids did not stimulate the rate of reaction. Phospholipid methylation was inhibited by S-adenosyl-l-homocysteine and by local anaesthetic drugs such as chlorpromazine and tetracaine which partition into the lipid bilayer. Control experiments demonstrated that the surface membrane-associated methyltransferase activity was not due to contamination of surface membrane preparations with intracellular membranes. Surface membranes were found to have higher specific methyltransferase activities than whole L-cell homogenates or endoplasmic reticulum-enriched microsomes. The low rate of methyltransferase function expressed in vitro (approximately 1 pmol/min · mg protein) suggests that phospholipid methylation is not a major metabolic source of surface membrane phosphatidylcholine.     

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.     

Phospholipid methylation in MOPC-31C cell membranes with modified phospholipid composition

The presence of the methylation pathway from phosphatidylethanolamine to phosphatidylcholine was first shown in MOPC-31C cells. Intermediate phospholipids of this pathway, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N′-dimethylethanolamine, were accumulated in the cell membranes by adding choline analogues such as N-monomethylethanolamine and N,N′-dimethylethanolamine to the culture medium. These modified membranes had a striking character of enhanced phospholipid methylation. This enhancement could be explained by increases in the second and the third step of the methylation pathway from phosphatidylethanolamine.     

Properties of Particulate and Detergent-Solubilized Phospholipid N-Methyltransferase Activity from Calf Brain

Abstract: Calf brain membranes catalyze the enzymatic transfer of [CH₃-³H]methyl groups from S-adenosyl-l -[CH₃-³H]methionine into endogenous phosphatidyl-N-methylethanolamine (PME), phosphatidyl-N,N-dimethylethanolamine (PDE), and phosphatidylcholine (PC). Phospholipid N-methylation can be stimulated by the addition of exogenous PME or PDE, added in aqueous dispersions with sodium taurocholate. When membranes are incubated in the presence of exogenous PME, [CH₃-³H]PDE represents 86% of the labeled phospholipid products. When exogenous PME is replaced by PDE, 91% of the label is incorporated into PC. Thus, under these in vitro conditions it is possible to assay PME- and PDE-N-methyitransferase activity separately. The calf brain phospholipid N-methyltransferase activity has also been solubilized by treating the membranes ultrasonically in the presence of Triton X-100 and 10 mM monothioglycerol. When the detergent extracts are incubated in the presence of exogenous PME, [CH₃-³H]PDE represents 86% of the enzymatically labeled products. In the presence of exogenous PDE, more than 97% of the label is incorporated into PC. Optimal conditions for the membrane-bound and detergent-solubilized PME- and PDE-N-methyltransferase activity have been established. These conditions have been used as a basis for testing the hypothesis that the conversion of PME to PC is catalyzed by a single enzyme in calf brain. In these studies, PME- and PDE-N-methyltransferase activities have been found to be similar, if not identical, with respect to: (1) extractability with Triton X-100; (2) pH optimum; (3) response to divalent cations; (4) apparent K_m, for S-adenosyl-l -methionine and K_I for S-adenosyl-l -homocysteine, (5) sensitivity to N-ethylmaleimide; and (6) thermal inactivation at 55°. Overall, these results are consistent with the conclusion that in calf brain, PME and PDE are methylated by the same enzyme or by two phospholipid N-methyltransferases having very similar properties.     

Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducer including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polycrylamide slab gel electrphoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with l-[¹⁴C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells.P180 was solubilized from ¹²⁵I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Modification of phospholipid metabolism in dibutyryl-cAMP-mediated morphological conversion of CHO cells.

The cellular uptake and incorporation of [1-¹⁴C]palmitic acid and ³²P into lipids of Chinese hamster ovary cells, clone K₁ (CHO-K₁) have been investigated under conditions where the cells are converted from the compact, epithelial-like shape to the elongated fibroblast-like morphology by N⁶,O^2′-dibutyryl adenosine 3′:5′-phosphate. The primary alteration in lipid metabolism accompanying the morphological conversion to the fibroblast form was an increased incorporation of lipid precursors into all phospholipid classes and a decreased incorporation into the “neutral” lipid fraction. These results reflect the cells' need for phospholipid precursors when the membrane expands to form the fibroblast shape. When the fibroblast-shaped cells were allowed to revert to the epithelial shape, lipid metabolism was similar to that found in untreated cells.     

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [¹⁴C-CH₃]-S-adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N-methylethanolamine, N,N-dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N-methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [³²P]-phosphatidyldimethylethanolamine to [³²P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually.Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.     

sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Mixed micellar analysis of the CPT1 and EPT1 gene products.

The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding distinct sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases. A haploid cpt1 ept1 double null mutant lacked detectable choline- and ethanolaminephosphotransferase activity but was viable for growth, establishing that these enzymes are nonessential. The activities of the CPT1 and EPT1 gene products were independently studied in membranes prepared from strains mutant in the cognate locus using mixed micellar assays. Both enzymes absolutely required phospholipid cofactors; half-maximal activation was observed at low mole fractions, suggesting that a small number of phospholipid molecules are required. The activities of the CPT1 and EPT1 gene products were compared with respect to dioleoylglycerol dependence, CDP-aminoalcohol specificity, phospholipid activation, and inhibition by CMP. The EPT1 gene product utilized CDP-ethanolamine, -monomethylethanolamine, -dimethylethanolamine, and -choline to significant extents, while the CPT1 gene product manifested relative specificity for CDP-choline and -dimethylethanolamine. The CPT1 and EPT1 gene products exhibited differing properties with respect to phospholipid activation, but this difference was dependent on the CDP-aminoalcohol substrate. In contrast, the two enzymes could be distinguished on the basis of their dioleoylglycerol dependencies, activation by Mg2+, and CMP inhibition profiles regardless of the CDP-aminoalcohol substrate employed. These studies provide the first definitive kinetic properties of individual choline- and ethanolaminephosphotransferases.     

Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccbaromyces cerevisiae

An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5′-CATGTGAAAT-3′) was found to be mandatory for CTR/HNM1 expression. This ‘decamer’ motif is located between nucleotides ?262 and ?271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of β-galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions ?213 or ?152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.     

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V_max values from 1,973 ± 118 to 380 ± 18 nmol·mg^–1·min^–1, when the binding affinity did not change significantly (K_m values 56 ± 8 and 53 ± 6 µM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages. protein trafficking     

Effects of Ganglioside GM3 on Phospholipid Turnover of Human Leukemic J6-2 Cells

Ganglioside GM₃ was reported to induce the differentiation of HL-60 cells to differentiate along the macrophage-monocytic route. We used human monocytoid leukemia J6-2 cells and successfully induced differentiation by GM₃. Because differentiation is accompanied by retarded growth rate and cell cycle is intimately related to phospholipid metabolism, so we explored how GM₃ was related to phospholipid metabolism. By using [³²P]Pi, [³H-CH₃]choline, [³H-CH₃]SAM, and [³H]inositol as radioactive tracers, we studied the turnover changes of phospholipids and their metabolites induced by GM₃. For the morphological changes of differentiation to occur, the cells had to be treated with GM₃ at a concentration of 50 M for 5-6 days, but the phospholipid changes occurred at a very early stage of GM₃ treatment (only 1 h). Our results indicate that GM₃ stimulated PE methylation pathway inhibited both CDP-choline pathway and PI cycle. The phospholipid changes may constitute the early events in differentiation induced by GM₃.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphospatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.     

Changes in phospholipid composition during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells(M1) could be induced by various inducers to form Fc receptors, phagocytize, migrate in agar, produce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. When M1 cells were cultured with inducer, the ratio of the percentage of phosphatidylethanolamine to that of phosphatidylcholine was increased about 2-fold. This ratio of the differentiated M1 cells was similar to that of peritoneal macrophages of normal mice or Mm-1 cells, which were established from spontaneously differentiated macrophage-like cells from M1 cells. These changes in phospholipid may be involved in the mechanisms of expression of the differentiation-associated phenotypic properties.     

Effect of Nutrients on Physiological Properties of Clostridium botulinum Type E

Eight strains of Clostridium botulinum type E out of twelve tested showed good growth and normal cell morphology in a synthetic medium containing choline. Growth and toxin production by a representative strain was not influenced by repeated subculturing. In the chemically defined medium, acetylcholine, N,N-dimethylethanolamine, and lecithin could replace choline to get normal cell division and cell morphology of C. botulinum type E. Choline could not be replaced by ethanolamine, N-methylethanolamine, or betaine. A toxigenic strain of C. botulinum type E showed proteolytic, lipolytic, and lecithinase activity in complex media but not in a synthetic medium. On prolonged incubation in the high temperature range of growth, the toxicity of the culture filtrate decreased in a complex, but not in a synthetic medium. The implications of these findings are discussed.     

Synthesis and evaluation of (S,S)-N,N′-bis-[3-(2,2′,6,6′-tetramethylbenzhydryloxy)-2-hydroxy-propyl]-ethylenediamine (S2824) analogs with anti-tuberculosis activity

In order to identify new and potent candidate drugs to treat tuberculosis, a library of compounds was screened, and (S,S)-N,N′-bis-[3-(2,2′,6,6′-tetramethylbenzhydryloxy)-2-hydroxy-propyl]-ethylenediamine (S2824) was identified as a hit in the screen. This research discusses our efforts to synthesize and test 30 analogs of this hit for activity against Mycobacterium tuberculosis. Two compounds with homopiperazine ring possess high in vitro activity against drug sensitive and resistant M. tuberculosis with MICs 0.78–3.13 μg/mL (or 1.22–4.88 μM).     

Early changes in ornithine decarboxylase activity during friend leukemia cell differentiation

Agents such as dimethylsulfoxide, N,N′-dimethylformamide and bisacetyldiaminopentane that induce erythroid differentiation of Friend leukemia cells, cause a rapid increase in ornithine decarboxylase (EC 4.1.1.17) activity in intact cells during the ‘latent’ period preceding the accumulation of hemoglobin-containing cells. Blockage of erythroid differentiation with 5-bromo-2′-deoxyuridine did not prevent these alterations in enzyme activity. Addition of each chemical inducer in the extracts of these cells stimulate the basal levels of ornithine decarboxylase activity. These data indicate that the chemical inducers of differentiation modify the normal pattern of ornithine decarboxylase activity in this system.     

Choline sulfokinase of Penicillium chrysogenum: partial purification and kinetic mechanism.

Choline sulfokinase (3′-phosphoadenosine 5′-phosphosulfate (PAPS):choline sulfotransferase, EC 2.8.2.6) was purified approximately 30-fold from the mycelium of Penicillium chrysogenum. The K_m for PAPS is 12 μm. The enzyme is remarkably specific for the adenosine 3′,5′ (or 2′-5′)-diphosphate moiety. 3′,5′-ADP (PAP) has a K_i of 2.5 to 14 μm (depending on the choline concentration) whereas the K_i values of 3′-AMP, 5′-AMP, and 5′-ADP are at least 300-fold higher. The enzyme is also highly specific for choline (K_m = 17 μM). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate (K_m = 800 μmV = 35% of V with choline). Triethylaminoethanol was a significantly poorer substrate (K_m = 2800 μM; V = 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the maximum activity decreases and the K_m for choline increases. (The K_m for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K_m for choline) by a 10-min preincubation with 50 mm mercaptoethanol. However, prolonged incubation (24 h) with 50 mm mercaptoethanol results in irreversible denaturation. Initial velocity studies established that the enzyme follows a sequential kinetic mechanism. Product inhibition studies suggest a rapid equilibrium random binding sequence. Choline-O-phosphate (a dead-end inhibitor) is linearly competitive with choline and a linear mixed type inhibitor with respect to PAPS. Choline analogs lacking the alcohol (or ester) group (e.g., trimethylammonium, neurine, chlorocholine) are competitive dead-end inhibitors with respect to choline but are uncompetitive with respect to PAPS. Thiocholine is a linear mixed type inhibitor with respect to PAPS, but the reciprocal plots are almost parallel. These results suggest that the analogs lacking an oxygen atom have a negligible affinity for the free enzyme and bind predominantly to the enzyme-PAPS complex.     

Choline and N,N-Dimethylethanolamine as Direct Substrates for Methanogens

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.     

Calcimycin mediates mycobacterial killing by inducing intracellular calcium-regulated autophagy in a P2RX7 dependent manner

Phenotypic screening led to the identification of calcimycin as a potent inhibitor of Mycobacterium bovis BCG (M. bovis BCG) growth in vitro and in THP-1 cells. In the present study, we aim to decipher the mechanism of antimycobacterial activity of calcimycin. We noticed that treatment with calcimycin led to up-regulation of different autophagy markers like Beclin-1, autophagy-related gene (Atg) 7, Atg 3 and enhanced microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) to LC3-II conversion in macrophages. This calcimycin-mediated killing of intracellular M. smegmatis and M. bovis BCG was abrogated in the presence of 3-methyladenine (3-MA). We also demonstrate that calcimycin binding with purinergic receptor P2X7 (P2RX7) led to increase in intracellular calcium level that regulates the extracellular release of ATP. ATP was able to regulate calcimycin-induced autophagy through P2RX7 in an autocrine fashion. Blocking of either P2RX7 expression by 1-[N,O-bis(5-Isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) or reducing intracellular calcium levels by 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) abrogated the antimycobacterial activity of calcimycin. Taken together, these results showed that calcimycin exerts its antimycobacterial effect by regulating intracellular calcium-dependent ATP release that induces autophagy in a P2RX7 dependent manner.