Phosphatidylcholine Analogues Research Articles

Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine.

Surface activity and sensitivity to inhibition from phospholipase A2 (PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions <1 mN/m under dynamic compression on the Wilhelmy balance and on a pulsating bubble surfactometer (37 degrees C, 20 cycles/min, 50% area compression). DEPN-8 + 1.5% SP-B/C exhibited even greater adsorption and had overall dynamic surface tension lowering equal to CLSE on the bubble. In addition, films of DEPN-8 + 1.5% SP-B/C on the Wilhelmy balance had better respreading than CLSE after seven (but not two) cycles of compression-expansion at 23 degrees C. DEPN-8 is structurally resistant to degradation by PLA2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2 led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.

Rapid transport of phospholipids across the plasma membrane of Leishmania infantum

The internalization of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) in Leishmania infantum promastigotes was studied. We observed a rapid inward redistribution of NBD-PC, -PE, and -PS across the plasma membrane at 28 and 4 °C. This internalization was shown to be independent of the endocytic activity of parasites. Rapid inward movement was coupled to an energy-dependent transporter because it was almost inhibited by depletion of cellular ATP and was blocked after pretreatment with N-ethylmaleimide (NEM). In contrast, NBD-SM traverses the plasma membrane by passive flip-flop. By comparing this pattern of phospholipid transbilayer movement with those known from other eukaryotic cells, candidate lipid transporters are discussed.

Selective caveolin-1-dependent endocytosis of glycosphingolipids.

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.

Bezafibrate, a second-generation fibrate analog, stimulates transport and secretion of phosphatidylcholine (PC) in human hepatocytes by a novel mechanism of redistribution of multidrug-resistance 3 P-glycoprotein (MDR3 Pgp) in the bile canaliculi

Bezafibrate, a second-generation fibrate analog, stimulates transport and secretion of phosphatidylcholine (PC) in human hepatocytes by a novel mechanism of redistribution of multidrug-resistance 3 P-glycoprotein (MDR3 Pgp) in the bile canaliculi

Model linear and star-shaped polyisoprenes with phosphatidylcholine analogous end-groups. Synthesis and association behavior in cyclohexane

Semitelechelic (ω-), telechelic (α,ω-), as well as 3- and 12-arm star polyisoprenes (PI), end-functionalized with the same dimethylamino group, were synthesized by anionic polymerization high vacuum techniques, using 3-dimethylaminopropyllithium as initiator and chlorosilanes as linking agents. The dimethylamino groups were transformed to phosphatidylcholine analogues through reaction with 2-ethoxy-2-oxo-1,3,2-dioxaphospholane. The association behavior of the phosphoro-zwitterionic PI (PZw-PI) was studied in cyclohexane by low angle laser light scattering, viscometry, and dynamic light scattering. The linear PZw-PIs present relatively low weight-average degree of association (Nw: 2–6), as compared with the corresponding sulfo-zwitterionic PI (Nw: 10–45). The Nw values decrease with increasing molecular weight of the PI. In the case of the star PZw-PI, the multiple intermolecular associations lead to the formation of gels, which break down by addition of a small amount of 1-heptanol. In very dilute solutions and for high arm molecular weight stars, intramolecular dominates over intermolecular association.

A Novel Membrane Protein, Ros3p, Is Required for Phospholipid Translocation across the Plasma Membrane inSaccharomyces cerevisiae

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.

Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells.

We studied the transport of the fluorescent cholesterol analog dehydroergosterol (DHE) in polarized HepG2 human hepatoma cells. DHE delivered via methyl-beta-cyclodextrin was delivered to both the apical and basolateral membranes and became concentrated in the apical membrane within 1 min. Intracellular DHE was targeted mainly to vesicles of the subapical compartment or apical recycling compartment (SAC/ARC), where it colocalized with fluorescent transferrin and fluorescent analogs of phosphatidylcholine and sphingomyelin. In contrast, transport of DHE from the plasma membrane to the trans-Golgi network was found to be very low. Vesicles containing DHE traversed the cells in both directions, but vesicular export of DHE from the SAC/ARC to the plasma membrane domains was low. Disruption of the microtubule cytoskeleton disturbed vesicular transport of DHE but not its enrichment in the apical (canalicular) membrane. Transport of DHE to the canalicular membrane after photobleaching was very rapid (t(12) = 1.6 min) and was largely ATP-independent in contrast to enrichment of DHE in the SAC/ARC. Release of DHE from the canalicular membrane was also ATP-independent but slower than the enrichment of sterol in the biliary canaliculus (t(12) = 5.4 min). Canalicular DHE could completely redistribute to the basolateral plasma membrane but could not transfer from one cell to the other cell of an HepG2 couplet. We conclude that sterol shuttles rapidly among the plasma membrane domains and other membrane organelles and that this nonvesicular pathway includes fast transbilayer migration.

Influence of the bovine seminal plasma protein PDC-109 on cholesterol in the presence of phospholipids.

The interaction of the major bovine seminal plasma protein PDC-109 with cholesterol was studied by employing spin-labelled analogues. It could be shown that PDC-109 does not interact directly with cholesterol molecules. However, in the presence of phospholipids we found a strong reduction of cholesterol motion by PDC-109. The fraction of immobilized cholesterol was largest for phosphorylcholine-containing lipids. This is consistent with the preferential interaction between PDC-109 and phosphatidylcholine. It is concluded that a stronger association and interaction of PDC-109 with phosphatidylcholine leads to an enhanced fraction of immobilized cholesterol analogues, but not to a phospholipid-dependent specific interaction between the protein and cholesterol. Moreover, the interaction of PDC-109 with various spin-labelled analogues of phosphatidylcholine (lysoPC, diacylPC) was investigated. In membranes of lipid vesicles the protein caused an immobilization of the phosphatidylcholine analogues mainly in the outer membrane leaflet, with no differences between diacylPC and lysoPC. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.

Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria.

In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.

Identification, Purification, and Characterization of a Thermally Stable Lipase from Rice Bran. A New Member of the (Phospho) Lipase Family

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 micromol min(-1) mg(-1) at 80 degrees C using [9,10-(3)H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80 degrees C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90 degrees C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76 degrees C and enthalpy 1.3 x 10(5) Calorie mol(-1) at this temperature. The purified lipase also exhibited phospholipase A(2) activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K(m) for triolein (6.71 mM) was higher than that for PC (1.02 mM). The enzyme preferentially hydrolyzed the sn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.

Identification, Purification, and Characterization of a Thermally Stable Lipase from Rice Bran. A New Member of the (Phospho) Lipase Family

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 μmol min−1 mg−1 at 80°C using [9,10-3H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80°C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90°C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76°C and enthalpy 1.3 × 105 Calorie mol−1 at this temperature. The purified lipase also exhibited phospholipase A2 activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K m for triolein (6.71 mm) was higher than that for PC (1.02 mm). The enzyme preferentially hydrolyzed thesn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.

Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography

Using electrospray ionization tandem mass spectrometry (ESI-MS/MS) this study shows that the loss of glycerophospholipid (GPL) after chromatography was unevenly distributed across the GPL molecular species. Both TLC and HPLC caused a preferential loss of GPL with 0 to 3 double bonds: 20% and 7.2% for choline glycerophosphates (PC) and 19.7% and 7.5% for ethanolamine glycerophosphates (PE), respectively. A consequence of these losses was that GPLs containing fatty acids with four or more double bonds had a greater contribution to the total after chromatography. ESI-MS/MS analysis also showed that PC molecular species with four or more double bonds migrated at the front of the TLC band of PCs. GPLs extracted from TLC plates occasionally contained PCs that were smaller than those in the original extract. These low molecular mass PCs were easily reduced to alcohols and formed derivatives with 2,4-dinitrophenylhydrazine, suggesting that aldehydes were generated by the oxidation of unsaturated fatty acids. Directly analyzing lipid extracts by ESI-MS/MS without preliminary chromatographic separation gives an accurate distribution of GPL molecular species in lipid mixtures. However, the ionization of the phospholipids in the electrospray jet maximized at relatively low concentrations of GPL. There was a linear response between phospholipid mass and ion intensity for concentrations around 1-2 nmol/ml for both PC and PE. The total ion intensity continued to increase with concentrations above 1-2 nmol/ml, but the response was non-linear.

Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator.

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.

Phospholipid scramblase activation pathways in lymphocytes.

In erythrocytes and platelets, activation of a nonspecific lipid flipsite termed the scramblase allows rapid, bidirectional transbilayer movement of all types of phospholipids. When applied to lymphoid cells, scramblase assays reveal a similar activity, with scrambling rates intermediate between those seen in platelets and erythrocytes. Scrambling activity initiated in lymphoid cells by elevation of intracellular Ca(2+) proceeds after a lag not noted in platelets or erythrocytes. The rates of transbilayer movement of phosphatidylserine and phosphatidylcholine analogues are similar whether the scramblase is activated by elevated internal Ca(2+) or by apoptosis. Elevation of internal Ca(2+) levels in apoptotic cells does not result in an additive increase in the rate of lipid movement. In lymphoid cells from a patient with Scott syndrome, scramblase cannot be activated by Ca(2+), but is induced normally during apoptosis. These findings suggest that Ca(2+) and apoptosis operate through different pathways to activate the same scramblase.

Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy.

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.

Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells.

We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and beta-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog beta-BODIPY-PC in the BC (t1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t1/2 = 18.5 min) at 37 degrees C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 degrees C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.

NBD‐Labeled Phosphatidylcholine and Phosphatidylethanolamine are Internalized by Transbilayer Transport across the Yeast Plasma Membrane

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.

Lipid class composition of the protozoan Perkinsus marinus, an oyster parasite, and its metabolism of a fluorescent phosphatidylcholine analog.

Perkinsus marinus is one of two important protozoan parasites of the eastern oyster, Crassostrea virginica. The other is Haplosporidium nelsoni. Lipids extracted from 7-d-old in vitro cultured P. marinus meronts, incubated with fluorescent-labeled phosphatidylcholine (FL PC) and nonincubated P. marinus meronts, were analyzed by a high-performance liquid chromatography (HPLC) system equipped with a diol phase column, in combination with thin-layer chromatography coupled with a flame-ionization detector (TLC/FID), and high-performance thin-layer chromatography (HPTLC). Various polar and neutral lipid classes were separated by HPLC using a two-gradient solvent system. Five polar lipid classes--phosphatidylcholine (PC), phosphatidylethanolamine (PE), cardiolipin (CL), sphingomyelin (SM), and phosphatidylserine (PS)--were identified from P. marinus extracts. Four neutral lipid classes--triacylglycerol (TAG), steryl ester (SE), cholesterol (CHO), and fatty alcohol--were distinguished. TLC/FID analysis of meront lipids showed that the weight percentages of PC, PE, CL, SM, PS/PI, TAG, SE, and CHO were 21, 10.7, 4, 2.3, 4.3, 48.7, 7.8, and 1.2%, respectively. HPLC and HPTLC analyses revealed the presence of two SM and PS isomers in P. marinus extracts. Perkinsus marinus effectively incorporated FL PC acquired from the medium and metabolized it to various components (i.e., free fatty acid, monoacylglycerol, diacylglycerol, TAG, PE, and CL). Uptake and interconversion of FL PC in P. marinus meronts increased with time. After 48 h the total uptake of fluorescence (FL) was 28.9% of the FL PC added to the medium, and 43% of the incorporated FL resided in TAG.

ChemInform Abstract: Synthesis of Novel Phenoxy-Linked Diazirine Glycero- and Sphingolipid Probes via Trialkylaryltin Intermediates.

Abstract Two photoaffinity lipid probes, phosphatidylcholine analog 1 and ceramide analog 2 , were synthesized via trialkylaryltin intermediates 10b , b ′ using a Stille coupling strategy. The latter are stable for months at −20°C. Iododestannylation afforded lipid probes 1 and 2 .