Phosphatidylcholine Derivatives Research Articles

Mechanisms of lipoplex formation: dependence of the biological properties of transfection complexes on formulation procedures.

Phospholipid-DNA complexes were made of the cationic triester derivative of phosphatidylcholine, EDOPC (1,2-dioleoyl- sn-glycero-3-ethylphosphocholine), by varying conditions of complex formation, in particular, the rate and direction of mixing, as well as by changing the mode of dispersing the lipid (extrusion or vortexing). The biological effects of variations in the formulation procedure were assessed by measuring transfection activity and cell association in cultures of BHK cells. Formulation procedures generally had little effect on cell association, but had marked effects on transfection efficiency. Transfection varied from effectively nil to extremely efficient with what appeared to be modest changes in formulation procedure. Formulation procedures also had significant effects on average sizes and size distributions of lipoplexes as determined by dynamic light scattering. Among the four possibilities of rapid or slow mixing combined with the two possible directions of mixing, slow addition of DNA to lipid gave results that differed significantly from the other three modes. In the case of vortexed lipid, the latter procedure was much less satisfactory than the other three, whereas in the case of extruded lipid, it was the only mode that produced satisfactory transfection. The factors that determine the difference in lipoplex properties can be identified as both geometric and physical. The geometric factor has to do with the symmetries of the participating units. There are three physical factors that are critical: the difference in vesicle stability upon interaction with DNA, the time dependence of interdiffusion of the components relative to that of vesicle rupture, and difference in input concentrations. These factors determine lipoplex size and, as already also shown by others, lipoplex size influences transfection efficiency.

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound ( S)-1-lyso-2-stearoylamino-2-deoxy- sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-κB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-κB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.

Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions.

Membrane lipids in most bacteria generally consist of the glycerophospholipids phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine (PE). A subset of bacteria also possesses the methylated derivatives of PE, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine (PC). In Sinorhizobium meliloti, which can form a nitrogen-fixing root nodule symbiosis with Medicago spp., PC can be formed by two entirely different biosynthetic pathways, either the PE methylation pathway or the recently discovered PC synthase pathway. In the latter pathway, one of the building blocks for PC formation, choline, is obtained from the eukaryotic host. Under phosphorus-limiting conditions of growth, S. meliloti replaces its membrane phospholipids by membrane-forming lipids that do not contain phosphorus; namely, the sulfolipid sulfoquinovosyl diacylglycerol, ornithine-derived lipids, and diacylglyceryl-N,N,N-trimethylhomoserine. Although none of these phosphorus-free lipids is essential for growth in culture media rich in phosphorus or for the symbiotic interaction with the legume host, they are expected to have major roles under free-living conditions in environments poor in accessible phosphorus. In contrast, sinorhizobial mutants deficient in PC show severe growth defects and are completely unable to form nodules on their host plants. Even bradyrhizobial mutants with reduced PC biosynthesis can form only root nodules displaying reduced rates of nitrogen fixation. Therefore, in the cases of these microsymbionts, the ability to form sufficient bacterial PC is crucial for a successful interplay with their host plants.

In-Situ ESR Detection of Radical Species of p-Benzoquinone in Aqueous Media

An in situ electrochemical ESR experiment was carried out to separately detect each reduction product of benzoquinone (BQ), anion radical (BQ−.) and radical (BQH.), in aqueous media. ESR spectra were obtained during the reduction of BQ at −0.3 V vs. Ag/AgCl in a pH range from 2.5 to 6.8. In situ ESR spectra exhibit two kinds of five narrowly separated hyperfine lines, which differ from the hyperfine structure in the line width (ΔH), the g value, and the line intensity ratio. At pHs greater than 3.1, a hyperfine structure corresponding to BQ−.was observed. On the other hands, a hyperfine structure corresponding to BQH. was observed at pHs between pH 3.0 and 3.1. The decomposition rate constants of BQ−. were also determined, and increased as the pH values of the media increased. In addition, in situ ESR spectra for the reduction reaction of BQ on phosphatidylcholine (PC) derivatives, such as dilauroyl-PC, dipalmitoyl-PC, and diarachidoyl-PC, were investigated in well-buffered solutions. The results showed that BQ−. was generated and stabilized in the lipid layers.

First synthesis of phosphatidylcholine and cholesteryl derivatives bearing an unsaturated aldehyde residue

The first synthesis of phosphatidylcholine and cholesteryl esters bearing a shortened acyl chain with a 2,4-dienal terminal is described.

Bromoacyl Analogs of Pyrene–Containing Phosphatidylcholine as New Fluorescent Lipid Probes with Intramolecular Quenching

Two new derivatives of phosphatidylcholine with an intramolecularly quenched fluorescence, namely, 1–ω–bromoundecanoyl–2–[4–(pyren–1–yl)butyroyl]–sn–glycero–3–phosphocholine (BPPhC) and 1–(9,10–dibromostearoyl)–2–[4–(pyren–1–yl)butyroyl]–sn–glycero–3–phosphocholine (DBPPhC), have been obtained by replacing acyl chains by residual pyrenebutyric and bromine–labeled fatty acids. Their structure has been verified by the 1H–NMR method and the spectral properties have been characterized in media of different polarity with the aid of absorption and fluorescent spectroscopy. It has been established that when BPPhC and DBPPhC are included in liposomes, the intensity of their fluorescence changes markedly as a function of the physical state of the bilayer, which makes it possible to use the compounds obtained as lipid probes for investigating the properties of biological and artificial membranes.

Thermophilic phospholipase A2 in the cytosolic fraction from the archaeon Pyrococcus horikoshii

AbstractHyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A2 in a cytosolic fraction. The enzyme displayed optimal activity at 90°C and pH 7 and preferentially hydrolyzed sn‐2 fatty acids in the following order: linoleoyl> oleoyl>arachidoyl phosphatidylcholine. Phospholipase A2 had similar activities toward l‐α‐1‐palmitoyl‐2‐arachidoyl derivatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A2 activity was unaffected by the addition of 0.0001–1 mM calcium, but was inhibited slightly by the addition of 2–10 mM calcium. The activity was enhanced more than 5–18‐fold in the presence of 3–20% (vol/vol) glycerol. The activity was unaffected by the addition of 1–5 mM EDTA or 0.01–20 mM dithiothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544 disappeared upon incubation of the cytosolic fraction with total lipid. The phospholipase A2 was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using 10–15% polyacrylamide gels containing sodium dodecyl sulfate (SDS‐PAGE). No activity of phospholipase A2 activity was observed in the absence of proteins less than 19 kD in size, as fractionated by SDS‐PAGE.

A HPLC–Fluorescence Detection Method for Determination of Cardiac Phospholipase D Activity in Vitro

A nonradioactive assay for the investigation of phospholipase D (PLD) activity in cardiac membranes has been developed. A fluorescent derivative of phosphatidylcholine [2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3proprionyl)amino) undecyl) sn-glycero-3-phosphocholine] was utilized as substrate in an in vitro PLD-catalyzed transphosphatidylation reaction utilizing ethanol as second substrate. Unreacted phosphatidylcholine and the products of phospholipase activity (PEtOH, phosphatidylethanol; PA, phosphatidic acid; DAG, diacylglycerol) were separated by a binary gradient HPLC system and detected by fluorometry. The detection limit of this assay is approximately 0.6 pmol PEtOH. The reaction proceeded at a linear rate for up to 45 min and increased linearly with increasing amounts of rat cardiac membrane protein in a range of 0.625 μg up to at least 25 μg. In the presence of potassium fluoride, formation of fluorescent PA increased at the expense of DAG generation, demonstrating the presence of PA phosphohydrolase activity in rat cardiac membranes. PEtOH formation was unchanged in the presence of the PA phosphohydrolase inhibitor, indicating that the phosphatidylalcohol is not subject to further metabolism by this enzyme. Activation of protein kinase C by phorbol ester significantly increased PLD activity in cardiac membranes. This assay proved to be sensitive for accurate and rapid assessment of PLD activity in cardiac membranes permitting further characterization of the regulation of PLD signal transduction in the heart.

Plasma membrane coating with cationic silica particles and osmotic shock alters the morphology of bovine aortic endothelial cells

We have used a published method of membrane preparation based on the precoating of the apical membrane of aortic endothelial cells with cationic silica microbeads (with or without polyacrylic acid) in combination with an osmotic shock and mechanical shearing to isolate the apical from the basal plasma membranes of these cells, in vitro. After labeling of the plasma membrane of adherent endothelial cells with a fluorescent derivative of phosphatidylcholine and by using laser confocal fluorescence scanning microscopy, we found that this method of membrane isolation rapidly induced invaginations of the basal plasma membrane to an extent which makes this method unsuitable for further membrane lipid analysis. Morphological analysis of the cells and fluorescence recovery after photobleaching experiments on the plasma membranes were performed at each step of the purification procedure and showed that only hypotonic shock and mechanical shearing of the cells enabled the basal plasma membranes to be purified without significant morphological changes.

Reduction of p-benzoquinone on lipid-modified electrodes: effect of the alkyl chain length of lipids on the electron transfer reactions

Using a phosphatidylcholine (PC) modified electrode, an unusual stabilization of p-benzoquinone anion radical (BQ−) is observed even in a buffered solution of pH 7.0. To clarify this incorporation reaction of BQ− in a PC layer, the effect of the molecular structure of the lipid was studied by constructing the PC modified electrodes with four PC derivatives; dicaproyl-, dilauroyl-, dipalmitoyl- and diarachidoyl-PC. At 25°C, dilauroyl-PC with a moderate alkyl chain length stabilized BQ− and allowed the approach of benzoquinol onto the surface of the modified electrode. From the pH dependence of the electrochemical responses, the incorporation mechanism of BQ− with P(O)OH was clarified. In addition to dilauroyl-PC, because dipalmitoyl-PC was found to incorporate BQ− over 40°C, the relationship between its incorporation and the phase transition temperature of each lipid has become clear. The correlation and the incorporation profiles were examined by measuring the oxidation of benzoquinol as well as that of BQ−. Also, the diffusion coefficients were determined for the lipids incorporating BQ−.

Solution conformations of short-chain phosphatidylcholine: Substrates of the phosphatidylcholine-preferring PLC of Bacillus cereus

The phosphatidylcholine (PC)-preferring phospholipase C (PLC) from Bacillus cereus (PLC Bc) hydrolyzes various 1,2-diacyl derivatives of PC at different rates. Substrates with side chains having eight or more carbons are present in micellular form in aqueous media and are processed most rapidly. The catalytic efficiency ( k cat/ K m) for the hydrolyses of short-chain PCs at concentrations below their respective critical micelle concentrations also decreases as the side chains become shorter, and this loss of efficiency owes its origin to increases in K m. In order to ascertain whether the observed increases in K m might arise from conformational changes in the glycerol backbone, nuclear magnetic resonance (NMR) experiments were performed in D 2O to determine the 3 J HH and 3 J CH coupling constants along the glycerol subunit of 1,2-dipropanoyl- sn-glycero-3-phosphocholine ( K m=61 mM), 1,2-dibutanoyl- sn-glycero-3-phosphocholine ( K m=21.2 mM) and 1,2-dihexanoyl- sn-glycero-3-phosphocholine ( K m=2.4 mM). Using these coupling constants, the fractional populations for each rotamer about the backbone of each of substrate were calculated. Two rotamers, which were approximately equally populated, about the sn-1– sn-2 bond of each substrate were significantly preferred, and in these conformers, the oxygens on the sn-1 and sn-2 carbons of the backbone were synclinal to optimize intramolecular hydrophobic interactions between the acyl side chains. There was greater flexibility about the sn-2– sn-3 bond, and each of the three possible staggered conformations was significantly populated, although there was a slight preference for the rotamer in which the oxygen bearing the phosphate head group was synclinal to the oxygen at the sn-2 carbon and to the sn-1 carbon; in this orientation, the head group is folded back relative to the side chains. These studies demonstrate that there is no significant change in the conformation about the glycerol backbone as a function of side chain length in short-chain phospholipids. Thus, prior organization of the substrate seems an unlikely determinant of the catalytic efficiency of PLC Bc, and other factors such as hydrophobic interactions or differential solvation/desolvation effects associated with the complexation of the substrate with PLC Bc may be involved.

Self-Assembly of Aromatic-Derivatized Amphiphiles: Phenyl, Biphenyl, and Terphenyl Fatty Acids and Phospholipids

This paper reports the synthesis of a series of amphiphiles (fatty acids and phosphatidylcholine derivatives) containing phenyl, biphenyl, and terphenyl chromophores inserted in the hydrocarbon chain and a study of their self-assembly in Langmuir−Blodgett films and aqueous dispersions. As observed and reported earlier for amphiphiles containing trans-stilbene, styrylthiophene, or azobenzene chromophores, several of the biphenyl and terphenyl derivatives show strong evidence of ground state association to form “H” aggregates characterized by a blue shift in absorption and a structured, red-shifted fluorescence. The phenyl amphiphiles show different behavior, suggesting that, even for pure films or bilayers, there is very little or no ground state association. For the biphenyl and terphenyl phospholipids, aqueous suspensions obtained by sonication are closed bilayer vesicles similar in size to those formed from the corresponding saturated phospholipids. The overall results of the present study indicate that...

Lecithinized superoxide dismutase induces vasodilation; evidence of direct contribution of superoxide anions to modulating vascular tone

The purpose of this study was to examine the role of superoxide anions in modulating the vascular tone. The effects of unmodified and lecithinized superoxide dismutase (SOD) on vascular tone were determined in aortic ring preparations of mice. In lecithinized SOD, 4 molecules of a phosphatidylcholine derivative were covalently bound to each dimer of recombinant human copper-zinc SOD to facilitate tissue accumulation. Unmodified SOD did not change vascular tone. However, lecithinized SOD induced dose-dependent vasodilation of aortic ring preparations. The pretreatment with N G-nitro-L-arginine methylester (L-NAME) 10 −4 mol/L abolished the vasodilation induced by lecithinized SOD. The results of this study indicate that superoxide anions play a prominent role in modulating the vascular tone by enhancing the breakdown of nitric oxide.

Ripple phase formation in phosphatidylcholine: Effect of acyl chain relative length, position, and unsaturation

The ripple phases of a range of mixed-acid phosphatidylcholine derivatives with one saturated C18 chain and the other chain of variable length or unsaturation were studied using differential scanning calorimetry, x-ray diffraction, and freeze-fracture electron microscopy. The temperature dependences of their ripple wavelength ~l!, stacking repeat distance ( ds), and the monoclinic angle u m defining their unit cell were measured and found to be consistent with an inherently asymmetric ripple phase with an amplitude that slowly increases with temperature. The temperature spans of the ripple phases of the saturated mixed-chain derivatives and the derivatives containing unsaturated chains were found to be larger and smaller, respectively, than those of homoacyl derivatives of the same equivalent chain length. This was shown to be consistent with the slidingchain model proposed by Cevc ~Biochemistry 30, 7186‐7197, 1991!. The tendency of phosphatidylcholine derivatives to form asymmetric ripple phases and the possible molecular organization of such phases are discussed in terms of different models. X-ray diffraction evidence was found for the existence of a secondary ripple phase with a wavelength about 1.8 times greater than the conventional pretransition ripple phase. This phase is formed in samples freshly cooled from the liquid-crystal phase and tends to persist longer than the conventional ripple phase on cooling to the gel phase. Freeze-fracture electron microscopy suggests that it is symmetric in cross section possibly reflecting the interaction of two opposing pretransition ripples. @S1063-651X~98!12209-2#

Oxidation of Dilinoleoyl Phosphatidylcholine by Lipoxygenase 1 from Soybeans

Soybean lipoxygenase-1 is able to oxidize dilinoleoyl phosphatidylcholine at pH 7.5 and 10. The reaction could be followed spectrophotometrically from the increase of the absorbance at 234 nm. An intermediate product and a final product were detected. In the intermediate product only one of the linoleoyl chains (eithersn1orsn2) was oxidized. In the final product, both linoleic acid units were converted into hydroperoxides. Apparently, oxidation of one of the linoleoyl chains leads to a disruption of the structure of the mixed bilayer disk, making the remaining fatty acid unit more accessible to the action of the enzyme. The specificity of lipoxygenase-1 when acting on phospholipids is not affected by pH. The exclusive production of 13-hydroperoxyoctadecadienoic acid derivatives of dilinoleoyl phosphatidylcholine at pH 7.5 and 10 may result from the blockage of the carboxylic end of the fatty acid.

Self-Assembly of Styrylthiophene Amphiphiles in Aqueous Dispersions and Interfacial Films: Aggregate Structure, Assembly Properties, Photochemistry, and Photophysics

Fatty acids and phosphatidyl choline derivatives incorporating the photoreactive trans-styrylthiophene chromophore have been prepared and studied in Langmuir−Blodgett films and aqueous dispersions, respectively. Both absorption and fluorescence spectra in the assemblies show prominent shifts indicative of aggregation similar to that observed with similar trans-stilbene and trans-azobenzene derivatives previously investigated. Studies of aqueous dispersions of the pure styrylthiophene phospholipids indicate the formation of structures much larger than the typical small unilamellar bilayer vesicles formed from saturated phospholipids of comparable chain length. Studies of the disaggregation process for two of the phospholipids give aggregation numbers of approximately 2 and 6, corresponding to 4 and 12 styrylthiophene units per aggregate. The stability of these aggregates is very similar to those of corresponding stilbene aggregates, and on the basis of spectral similarities it seems reasonable to propose a “pinwheel” fraction of a glide or herringbone structure as the “unit aggregate” and predominate species present in the aqueous dispersions. However, the chief photoreaction observed for the aqueous dispersions of the phospholipids is dimerization to form the syn head-to-head dimer, consistent with topologically controlled reaction from a translation structure in which nearest-neighbor chromophores are aligned parallel. Simulations suggest that while a translation layer structure may be of lowest energy, glide layer structures that show good agreement between measured and predicted properties are also energetically accessible. A possible explanation for the observed reactivity and photophysics is that within the aqueous dispersions there may be equilibration between “glide” and “translation” structures. When mixtures of the styrylthiophene phospholipids and saturated phospholipids are codispersed, bilayer vesicles are formed that are capable of entrapping reagents such as carboxyfluorescein. Irradiation of the aggregated trans-styrylthiophene in these vesicles leads to release of the entrapped carboxyfluorescein concurrent with photodimerization of the styrylthiophene. The pattern of reagent release is similar to that observed upon photoisomerization of the corresponding azobenzene aggregates and suggests a “catastrophic” destruction of the vesicles.

Supramolecular Aggregates of Azobenzene Phospholipids and Related Compounds in Bilayer Assemblies and Other Microheterogeneous Media: Structure, Properties, and Photoreactivity1

A number of phosphatidyl choline derivatives containing trans-azobenzene units in the fatty ester backbone have been synthesized and studied in aqueous dispersions both pure and in the presence of saturated and unsaturated phospholipids. The structures of the assemblies formed have been investigated by microcalorimetry, dynamic light scattering, cryo-transmission electron microscopy, and reagent entrapment. While many of the mixed phospholipid dispersions give evidence for the formation of small unilamellar vesicles, the aqueous dispersions of pure azobenzene phospholipids (APL's) give evidence for several different structures, including relatively large plates in at least one case. The azobenzenes show strong evidence of “H” aggregate formation both in the pure and mixed dispersions. The aggregation number has been estimated for several of the APL's and found to be typically 3 or a multiple thereof. On the basis of simulations and studies with similar stilbene phospholipids as well as on the strong induced circular dichroism signals observed for the aggregate, we infer a chiral “pinwheel” unit aggregate structure similar to that found for several aromatics. The azobenzenes in the aqueous dispersions have been found to photoisomerize to give cis-rich photostationary states; the cis-azobenzenes show no evidence for aggregation and no induced circular dichroism. The cis-azobenzenes can be isomerized back to the trans either by irradiation or by thermal paths. Mixed aqueous dispersions of trans-APL's with saturated or unsaturated phospholipids can be prepared which entrap the fluorescent dye carboxyfluorescein (CF) under conditions where the CF fluorescence is very low due to self-quenching. By varying the APL/host phospholipid ratio the azobenzene can be aggregate, monomer, or dimer. In cases where the azobenzene is monomer or dimer, irradiation produces complete isomerization but little “leakage” of CF from the vesicle interior. In contrast, where the azobenzene is predominantly aggregate, irradiation results in both photoisomerization and reagent release. That photoisomerization in the latter case can result in “catastrophic” destruction of the vesicle can also be shown by cryo-transmission electron microscopy.

Interaction of Bee Venom Melittin with Zwitterionic and Negatively Charged Phospholipid Bilayers: A Spin-Label Electron Spin Resonance Study

Electron spin resonance (ESR) spectroscopy was used to study the penetration and interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) and ditetradecylphosphatidylglycerol (DTPG) bilayer membranes. Melittin is a surface-active, amphipathic peptide and serves as a useful model for a variety of membrane interactions, including those of presequences and signal peptides, as well as the charged subdomain of the cardiac regulatory protein phospholamban. Derivatives of phosphatidylcholine and phosphatidylglycerol spin-labeled at various positions along the sn-2 acyl chain were used to establish the chain flexibility gradient for the two membranes in the presence and absence of melittin. Negatively charged DTPG bilayer membranes showed a higher capacity for binding melittin without bilayer disruption than did membranes formed by the zwitterionic DMPC, demonstrating the electrostatic neutralization of bound melittin by DTPG. The temperature dependence of the ESR spectra showed that the gel-to-liquid crystalline phase transition is eliminated by binding melittin to DTPG bilayers, whereas a very broad transition remains in the case of DMPC bilayers. None of the spin labels used showed a two-component spectrum characteristic of a specific restriction of their chain motion by melittin, but the outer hyperfine splittings and effective chain order parameters were increased for all labels upon binding melittin. This indicates a reduced flexibility of the lipid chains induced by a surface orientation of the bound melittin. Whereas the characteristic shape of the chain flexibility gradient was maintained upon melittin addition to DMPC bilayers, the chain flexibility profile in DTPG bilayers was much more strongly perturbed. It was found that the steepest change in segmental flexibility was shifted toward the bilayer interior when melittin was bound to DTPG membranes, indicating a greater depth of penetration than in DMPC membranes. pH titration of stearic acid labeled at the C-5 position, used as a probe of interfacial interactions, showed net downward shifts in interfacial pK of 0.8 and 1.2 pH units contributed from the positive charge of melittin, outweighing upward shifts from interfacial dehydration, when melittin was bound to DTPG and DMPC, respectively. The perturbation of the outer hyperfine splitting was used to determine the interactions of melittin with spin-labeled lipids of different polar headgroups in DTPG and DMPC. Anionic lipids (phosphatidylserine, phosphatidylglycerol, and stearic acid) and zwitterionic lipids (phosphatidylethanolamine and phosphatidylcholine) had the largest outer splittings in the presence of melittin. Neutral lipids (protonated stearic acid and diacylglycerol) displayed the largest increase in outer splitting on binding melittin, which was attributed to a change in the vertical location of these lipids in the bilayer. Both effects were more pronounced in DTPG than in DMPC.

Digestion of phosphatidylcholines, absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro.

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphatidylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1- [1-14C]acyl- and 1-[3H] alkyl-lysophosphatidylcholine, [1-14C]oleic acid, [2-14C]glycerol, phosphoryl[methyl-14C]choline, and [methyl-14C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A2, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and Pi, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoacylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine.

Lecithinization of IL-6 enhances its thrombopoietic activity in mice.

This study was conducted to assess the merit of lecithinization of recombinant human interleukin-6 (IL-6) as a drug delivery system. IL-6 was lecithinized by covalently binding it with a phosphatidylcholine (lecithin, PC) derivative. The in-vivo thrombopoietic potency of lecithinized IL-6 (PC-IL-6) was greater than that of native IL-6 when administered subcutaneously, although the in-vitro bioactivity of PC-IL-6 was markedly reduced by lecithinization. When PC-IL-6 and native IL-6 were given in doses that produced the same level of thrombopoietic activity, the former stimulated less production of IgG1, a marker of the adverse effects of IL-6, than did the latter. Furthermore, PC-IL-6 persisted in the blood longer than native IL-6. Based on the above, PC-IL-6 appears to be useful as a drug delivery system and may also be useful in the treatment of drug-induced thrombocytopenia.