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Sphingosylphosphorylcholine (SPC) participates in several cellular processes including metastasis. SPC induces keratin reorganization and regulates the viscoelasticity of metastatic cancer cells including PANC-1 cancer cells leading to enhanced migration and invasion. The role of SPC and the relevant mechanism in invasion of breast cell are as yet unknown. SPC dose-dependently induces invasion of breast cancer cells or breast immortalized cells. Reverse transcription polymerase chain reaction and Western blot analyses of MCF10A and ZR-75-1 cells indicated that SPC induces expression and secretion of matrix metalloproteinase-3 (MMP3). From online KMPLOT, relapse free survival is high in patients having low MMP3 expressed basal breast cancer (n=581, p=0.032). UK370106 (MMP3 inhibitor) or gene silencing of MMP3 markedly inhibited the SPC-induced invasion of MCF10A cells. An extracellular signal-regulated kinase (ERK) inhibitor, PD98059, significantly suppressed the secretion and the gelatinolytic activity of MMP3, and invasion in MCF10A cells. Over-expression of ERK1 and ERK2 promoted both the expression and secretion of MMP3. In contrast, gene silencing of ERK1 and ERK2 attenuated the secretion of MMP3 in MCF10A cells. The effects of SPC-induced MMP3 secretion on β-catenin and TCF/lymphoid enhancer factor (LEF) promoter activity were examined since MMP3 indirectly activates canonical Wnt signaling. SPC induced translocation of β-catenin to nucleus and increased TCF/LEF promoter activity. These events were suppressed by UK370106 or PD98059. Wnt inhibitor, FH535 inhibited SPC-induced MMP3 secretion and invasion. Taken together, these results suggest that SPC induces MMP3 expression and secretion via ERK leading to Wnt activation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Resident cardiac Sca-1-positive (+) stem cells may differentiate into cardiomyocytes to improve the function of damaged hearts. However, little is known about the inducers and molecular mechanisms underlying the myogenic conversion of Sca-1(+) stem cells. Here we report that sphingosylphosphorylcholine (SPC), a naturally occurring bioactive lipid, induces the myogenic conversion of Sca-1(+) stem cells, as evidenced by the increased expression of cardiac transcription factors (Nkx2.5 and GATA4), structural proteins (cardiac Troponin T), transcriptional enhancer (Mef2c) and GATA4 nucleus translocation. First, SPC activated JNK and STAT3, and the JNK inhibitor SP600125 or STAT3 inhibitor stattic impaired the SPC-induced expression of cardiac transcription factors and GATA4 nucleus translocation, which suggests that JNK and STAT3 participated in SPC-promoted cardiac differentiation. Moreover, STAT3 activation was inhibited by SP600125, whereas JNK was inhibited by β-cyclodextrin as a lipid raft breaker, which indicates a lipid raft/JNK/STAT3 pathway involved in SPC-induced myogenic transition. β-Catenin, degraded by activated GSK3β, was inhibited by SPC. Furthermore, GSK3β inhibitors weakened but the β-catenin inhibitor promoted SPC-induced differentiation. We found no crosstalk between the lipid raft/JNK/STAT3 and β-catenin pathway. Our study describes a lipid, SPC, as an endogenic inducer of myogenic conversion in Sca-1(+) stem cells with low toxicity and high efficiency for uptake., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Miltefosine possesses antiparasitic, antibacterial, antifungal and antitumor activities; however, its mechanism of action is not well established. In the current work, the miltefosine concentrations required to achieve 50% hemolysis in PBS were shown to vary from 600 μM using 5×10(9) cells/mL to ~2.9 μM for ~5×10(6) cells/mL. This cell concentration-dependent hemolytic potential was described using an equation that included the membrane-water partition coefficient (K) and miltefosine concentrations in the cell membrane (cm) and aqueous medium (cw) as variables. The best-fit values for the 50% hemolysis data were log K=4.68, cm=110.8 mM, and cw=2.3 μM. Hemolysis measurements in whole blood were used to determine the erythrocyte membrane-plasma partition coefficient of miltefosine (Log KM/P=1.77). Additionally, miltefosine concentration in whole blood was found to be ~86% of that in plasma. Previously reported clinical pharmacokinetics data indicate that the plasma concentration of miltefosine peaks at ~90 μg/mL when treating visceral leishmaniasis. Using this concentration, which corresponds to ~77 μg/mL miltefosine in whole blood, we found only 2.8% hemolysis. Significant hemolysis (5.4%) was observed only after doubling the concentration to 180 μg/mL. Recently reported data indicate that miltefosine inhibitory concentrations in Leishmania are also dependent on cell concentration. The biophysical parameters assessed in the current study indicated that this type of response is associated with the accumulation of the drug in the cell membrane, which becomes damaged when critical drug concentrations are reached., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear. We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells. Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Autophagy, evoked by diverse stresses including myocardial ischemia/reperfusion (I/R), profoundly affects the development of heart failure. However, the specific molecular basis of autophagy remains to be elucidated. Here we report that sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, significantly suppressed apoptosis and induced autophagy in cardiomyocytes. Blocking this SPC evoked autophagy by 3-methyladenine (3MA)-sensitized cardiomyocytes to serum deprivation-induced apoptosis. Subsequent studies revealed that SPC downregulated the phosphorylation of p70S6K and 4EBP1 (two substrates of mTOR) but enhanced that of JNK when inducing autophagy. We identified SPC as a switch for the activity of Akt1, a supposed upstream modulator of both mTOR and JNK. Furthermore, β-cyclodextrin, which destroys membrane cholesterol, abolished the SPC-reduced phosphorylation of both Akt and PTEN, thus inhibiting SPC-induced autophagy. In conclusion, SPC is a novel molecule protecting cardiomyocytes against apoptosis by promoting autophagy. The lipid raft/PTEN/Akt1/mTOR signal pathway is the underlying mechanism and might provide novel targets for cardiac failure therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Synthetic alkylphospholipids (ALPs), such as edelfosine, miltefosine, perifosine, erucylphosphocholine and erufosine, represent a relatively new class of structurally related antitumor agents that act on cell membranes rather than on DNA. They selectively target proliferating (tumor) cells, inducing growth arrest and apoptosis, and are potent sensitizers of conventional chemo- and radiotherapy. ALPs easily insert in the outer leaflet of the plasma membrane and cross the membrane via an ATP-dependent CDC50a-containing 'flippase' complex (in carcinoma cells), or are internalized by lipid raft-dependent endocytosis (in lymphoma/leukemic cells). ALPs resist catabolic degradation, therefore accumulate in the cell and interfere with lipid-dependent survival signaling pathways, notably PI3K-Akt and Raf-Erk1/2, and de novo phospholipid biosynthesis. At the same time, stress pathways (e.g. stress-activated protein kinase/JNK) are activated to promote apoptosis. In many preclinical and clinical studies, perifosine was the most effective ALP, mainly because it inhibits Akt activity potently and consistently, also in vivo. This property is successfully exploited clinically in highly malignant tumors, such as multiple myeloma and neuroblastoma, in which a tyrosine kinase receptor/Akt pathway is amplified. In such cases, perifosine therapy is most effective in combination with conventional anticancer regimens or with rapamycin-type mTOR inhibitors, and may overcome resistance to these agents. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Activation of the pro-apoptotic protein Bax under intracellular oxidative stress is closely related to its association with the mitochondrial outer membrane (MOM) system, ultimately resulting in cell death. The precise mechanism by which this activation and the subsequent structural changes in the protein occur is currently unknown. In addition to triggering the onset of apoptosis, oxidative stress generates oxidized lipids whose impact on mitochondrial membrane integrity and the activity of membrane-associated Bax is unclear. We therefore devised a model system that mimics oxidative stress conditions by incorporating oxidized phospholipids (OxPls) into mitochondria-like liposomes, and studied the OxPls' impact on Bax-membrane interactions. Differential scanning calorimetry (DSC) was used to study membrane organization and protein stability, while conformational changes in the protein upon contact with lipid vesicles were monitored using far-UV circular dichroism (CD) spectroscopy. The thermograms for liposomes containing the OxPl 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) differed dramatically from those for unmodified liposomes. Moreover, Bax exhibited enhanced thermal stability in the presence of the modified liposomes, indicating that it interacted strongly with PazePC-containing membranes. The presence of PazePC also increased the α-helical character of Bax compared to the protein alone or with PazePC-free vesicles, at 10°C, 20°C, and 37°C. Presumably, the presence of PazePC-like OxPls a) increases the population of membrane-associated Bax and b) facilitates the protein's insertion into the membrane by distorting the bilayer's organization, as seen by solid-state high-resolution (1)H and (31)P magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Structural changes for a series of antimicrobial peptides in various solvents were investigated by a combined approach of FTIR and CD spectroscopy. The well-characterized and potent antimicrobial peptides indolicidin and tritrpticin were studied along with several analogs of tritrpticin, including Tritrp1 (amidated analog of tritrpticin), Tritrp2 (analog of Tritrp1 with Arg-->Lys substitutions), Tritrp3 (analog of Tritrp1 with Pro-->Ala substitutions) and Tritrp4 (analog of Tritrp1 with Trp-->Tyr substitutions). All peptides were studied in aqueous buffer, ethanol and in the presence of dodecylphosphocholine (DPC) micelles. It was shown that tritrpticin and its analogs preferentially adopt turn structures in all solvents studied. The turn structures formed by the tritrpticin analogs bound to DPC micelles are more compact and more conformationally restricted compared to indolicidin. While several peptides showed a slight propensity for an alpha-helical conformation in ethanol, this trend was only strong for Tritrp3, which also adopted a largely alpha-helical structure with DPC micelles. Tritrp3 also demonstrated along with Tritrp1 the highest ability to interact with DPC micelles, while Tritrp2 and Tritrp4 showed the weakest interaction.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in a variety of cellular responses. In the present study, we demonstrated that treatment of human adipose tissue-derived mesenchymal stem cells (hATSCs) with D-erythro-SPC resulted in apoptosis-like cell death, as demonstrated by decreased cell viability, DNA strand breaks, the increase of sub-G1 fraction, cytochrome c release into cytosol, and activation of caspase-3. In contrast, the exposure of hATSCs to L-threo-SPC did not induce the cell death, suggesting that the SPC-induced cell death was selective for the D-erythro-stereoisomer of SPC. The D-erythro-SPC-induced cell death was prevented by DEVD-CHO, a caspase-3 specific inhibitor, and Z-VAD-FMK, a general caspase inhibitor, suggesting that the SPC-induced cell death of hATSCs occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, D-erythro-SPC treatment stimulated the activation of mitogen-activated protein kinases, such as ERK and c-Jun NH2-terminal protein kinase (JNK), and the D-erythro-SPC-induced cell death was completely prevented by pretreatment with the MEK inhibitor, U0126, but not by pretreatment with the JNK inhibitor, SP600125, and the p38 MAPK inhibitor, SB202190, suggesting a specific involvement of ERK in the D-erythro-SPC-induced cell death. Pretreatment with U0126 attenuated the D-erythro-SPC-induced release of cytochrome c. From these results, we suggest that ERK is involved in the SPC-induced cell death of hATSC through stimulation of the cytochrome c/caspase-3-dependent pathway.

The membrane-bound proton-pumping inorganic pyrophosphatase from Rhodospirillum rubrum was heterologously expressed in Escherichia coli C43(DE3) cells and was inhibited by 4-bromophenacyl bromide (BPB), N,N'-dicyclohexylcarbodiimid (DCCD), diethyl pyrocarbonate (DEPC) and fluorescein 5'-isothiocyanate (FITC). In each case, the enzyme activity was rather well protected against inhibitory action by the substrate Mg(2)PPi. Site-directed mutagenesis was employed in attempts to identify target residues for these inhibitors. D217 and K469 appear to be the prime targets for DCCD and FITC, respectively, and may thus be involved in substrate binding. No major effect on enzyme activities was seen when any one of the four histidine residues present in the enzyme were substituted. Nevertheless, a mutant with all of the four charged histidine residues replaced retained only less than 10% of the hydrolysis and proton-pumping activities. Substitution of D217 with A or H yielded an enzyme with at least an order of magnitude lower hydrolysis activity. In contrast with the wild-type, these variants showed higher hydrolysis rates at lower concentrations of Mg(2+), possibly reflecting a change in substrate preference from Mg(2)PPi to MgPPi. BPB is a H(+)-pyrophosphatase inhibitor that apparently has not been used previously as an inhibitor of these enzymes.

Hipposin is a potent 51-mer antimicrobial peptide (AMP) from Atlantic halibut with sequence similarity to parasin (19-mer catfish AMP), buforin I (39-mer toad AMP), and buforin II (an active 21-mer fragment of buforin I), suggesting that the antimicrobial activity of these peptides might all be due to a common antimicrobial sequence motif. In order to identify the putative sequence motif, the antimicrobial activity of hipposin fragments against 20 different bacteria was compared to the activity of hipposin, parasin and buforin II. Neither parasin nor the 19-mer parasin-like fragment HIP(1-19) (differs from parasin in only three residues) that is derived from the N-terminal part (residues 1-19) of hipposin had marked antimicrobial activity. In contrast, the fragment HIP(16-36) (identical to buforin II) that is derived from the middle part of hipposin (residues 16-36) had such activity, indicating that this part of hipposin contained an antimicrobial sequence motif. The activity was enhanced when the parasin-like N-terminal sequence was also present, as the fragment HIP(1-36) which consists of residues 1-36 in hipposin was more potent than HIP(16-36). Extending HIP(1-36) with three C-terminal residues-thereby constructing the buforin I-like peptide HIP(1-39) (differs from buforin I in only three residues)-increased the activity further. Also, the presence of the C-terminal part of hipposin (residues 40-51) increased the activity, as hipposin was clearly the most potent of all the peptides that were tested. Circular dichroism structural analysis of the peptides revealed that they were all non-structured in aqueous solution. However, trifluoroethanol and the membrane-mimicking entities dodecylphosphocholine micelles and negatively charged liposomes induced (amphiphilic) alpha-helical structuring in hipposin. Judging from the structuring of the individual fragments, the tendency for alpha-helical structuring appeared to be greater in the C-terminal and the buforin II-like middle region of hipposin than in the parasin-like N-terminal region.

The anticancer drug hexadecylphosphocholine (HePC), an alkyl-lysophospholipid analog (ALP), has been shown to induce apoptosis and inhibit the synthesis of phosphatidylcholine (PC) in a number of cell lines. We investigated whether inhibition of PC synthesis plays a major causative role in the induction of apoptosis by HePC. We therefore directly compared the apoptosis caused by HePC in CHO cells to the apoptotic process in CHO-MT58 cells, which contain a genetic defect in PC synthesis. HePC-provoked apoptosis was found to differ substantially from the apoptosis observed in MT58 cells, since it was (i) not accompanied by a large decrease in the amount of PC and diacylglycerol (DAG), (ii) not preceded by induction of the pro-apoptotic protein GADD153/CHOP, and (iii) not dependent on the synthesis of new proteins. Furthermore, lysoPC as well as lysophosphatidylethanolamine (lysoPE) could antagonize the apoptosis induced by HePC, whereas only lysoPC was able to rescue MT58 cells. HePC also induced a rapid externalisation of phosphatidylserine (PS). These observations suggest that inhibition of PC synthesis is not the primary pathway in HePC-induced apoptosis.

Phosphorylcholine (PC) is increasingly becoming recognised as a carbohydrate-associated component of a wide variety of procaryotic and eucaryotic pathogens. Studies employing nematode PC-containing molecules indicate that it possesses a plethora of immunomodulatory activities. ES-62 is a PC-containing glycoprotein, which is secreted by the rodent filarial nematode Acanthocheilonema viteae and which provides a model system for the dissection of the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitised humans, ES-62 is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as PC conjugated to albumin or even PC alone broadly mimic the results obtained with ES-62. PC-induced impaired lymphocyte responsiveness appears to reflect uncoupling of the antigen receptors from key intracellular proliferative signalling events such as the phosphoinositide 3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. Although PC-ES-62 can desensitise B and T cells, not all cells are affected, and in fact it is still possible to generate an antibody response to the molecule. Dissection of this response indicates that it is of the TH-2 type. This appears to reflect the ability of ES-62 to direct the polarity of the T cell response by suppressing the production of proinflammatory cytokines, inducing the induction of anti-inflammatory cytokines and by driving the maturation of dendritic cells that direct TH-2 T cell responses.

Liposomes from octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP), a new alkylphospholipid derivative with an improved cancerostatic activity, were prepared for the first time and the activity in vitro and in vivo was characterised. The formation of liposomes (MLV, SUV and LUVET) differing in cholesterol content, charge, and sterical stabilisation is possible without serious problems, despite the lysolipid-like structure of the OPP. Liposomes with a low amount of cholesterol and with PEG2000DSPE-coating were the most stable OPP liposomes, both in buffer and in serum. The cytotoxicity of micellar or liposomal OPP against breast cancer cell lines in vitro was in the range of 20-60 microM. The cytotoxicity of the liposomal formulation was inversely related to the content of cholesterol, whereas the sterical stabilisation and/or the incorporation of a positive charge had only a very moderate modulating effect on the inhibition of cell proliferation. The strongest antitumour effect on the xenotransplanted breast cancer MT-3 in vivo was obtained with sterically stabilised OPP liposomes with low CH content. The beneficial therapeutic effect of these liposomes was accompanied by better tolerance and a significant inhibition of haemolysis compared to micellar OPP.

We previously demonstrated that the sphingolipid, sphingosylphosphocholine (SPC) increased DNA binding activity of AP-1 proteins accompanying cellular proliferation. Herein, the effects of SPC on DNA binding activity and transcription of the basic, helix-loop-helix, leucine zipper (bHLH-ZIP) proteins Myc, Max, and USF were investigated because they regulate genes involved in mitogenesis. E-box (CACGTG) DNA binding proteins were detected by electrophoretic mobility shift assays in nuclear extracts from Swiss 3T3 fibroblasts. The slowest migrating complex (complex I) increased within 1-3 min after treatment with SPC, remained elevated for 10 min, and increased again after 12 h. Complexes I and II contained USF-1 and USF-2 proteins, and complex I migrated similarly to recombinant USF-1 protein/DNA complex. Treatment of nuclear extracts with alkaline phosphatase decreased these complexes suggesting USF might be a phosphoprotein, post-translationally modified by SPC. max and usf-1 mRNA levels were unaffected by SPC treatment. In contrast, c-myc mRNA was rapidly elevated, reached maximum levels at 0.5-1 h, and showed an additional increase after 12 h, just preceding S phase. Thus, certain bHLH-ZIP transcription factors may be involved in cell growth regulation by SPC., (Copyright 1998 Elsevier Science B.V.)

The mechanisms that account for the anti-proliferative properties of the biologically active lysophospholipid analog hexadecylphosphocholine (HexPC) were investigated in HL60 cells. HexPC inhibited the incorporation of choline into phosphatidylcholine and the pattern of accumulation of soluble choline-derived metabolites pinpointed CTP:phosphocholine cytidylyltransferase (CT) as the inhibited step in vivo. HexPC also inhibited recombinant CT in vitro. HexPC treatment led to accumulation of cells in G2/M phase, triggered DNA fragmentation and caused morphological changes associated with apoptosis. The supplementation of HexPC-treated cells with exogenous lysophosphatidylcholine (LPC) completely reversed the cytotoxic effects of HexPC and restored HL60 cell proliferation in the presence of the drug. LPC provided an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC. This result contrasted with the response of HL60 cells to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) where LPC overcame the cytotoxic effects but did not support continued cell proliferation. Morphological integrity, DNA stability and cell viability were maintained in cells treated with LPC plus either antineoplastic agent. Thus the inhibition of phosphatidylcholine biosynthesis at the CT step accounts for the cytotoxicity of both HexPC and ET-18-OCH3 which is overridden by providing an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC.

In a pancreatic duct adenocarcinoma cell line (CFPAC-1) sphingosine (10 microM) induced both mobilization of intracellular Ca2+ and stimulation of inositol phosphates accumulation. Whereas this latter effect was significantly inhibited by treatment with pertussis toxin or by short-term incubation with phorbol 12-myristate 13-acetate, Ca2+ mobilization was completely insensitive to both treatments. Experiments with permeabilized cells showed that sphingosine or the sphingosine metabolites sphingosine-1-phosphate and sphingosylphosphorylcholine were unable to directly release Ca2+ from internal stores, whereas phosphatidic acid, but not arachidonic acid, was effective. Phosphatidic acid formation was markedly enhanced (2.9-fold over control) by sphingosine, this effect being significantly reduced by preincubation with the diacylglycerol kinase inhibitor R59022. Ca2+ mobilization by sphingosine was also cut down by preincubation with R59022. In conclusion, the results suggest that sphingosine activates phospholipase C through a mechanism functionally coupled through a G protein and under control of PKC. Mobilization of [Ca2+]i by sphingosine is independent of phospholipase C stimulation and likely due to elevation of phosphatidic acid generated by stimulation of diacylglycerol kinase activity.

In exploring the occurrence and mechanism of liposome-encapsulated hemoglobin (LEH)-induced complement (C) activation, we found that normal human serum contained low titers of IgG and IgM class natural antibodies with reactivity against LEH, and that the amount of vesicle-bound IgM significantly correlated with LEH-induced C consumption. IgM binding to LEH was inhibited by phosphocholine and ATP, but not by choline chloride. These data suggest that naturally occurring antibodies play a key role in LEH-induced C activation, and that a major portion of these antibodies are directed against the phosphate moiety on the phospholipid headgroups of liposome bilayers.

The segment, YSDELRQRLAARLEALKENG, corresponding to residues 166 to 185 of human serum apolipoprotein A-I, was studied by circular dichroism and NMR spectroscopy in sodium dodecyl sulfate and dodecylphosphocholine micelles. 2-Dimensional NOESY, TOCSY and DQF-COSY spectra of apoA-I(166-185) in perdeuterated sodium dodecyl sulfate (SDS-d25) and dodecylphosphocholine (DPC-d38) micelles were collected at a peptide/SDS (DPC) ratio of 1:40. Similar CD spectra and NOE connectivity patterns were observed for apoA-I(166-185) in SDS and DPC, indicating a similar helical conformation in both. Conformations of apoA-I(166-185) in DPC-d38 micelles, and in SDS-d25 micelles at two pH values, 6.6 and 3.7, were determined using distance geometry calculations. Backbone superposition (N,C alpha,C = O) for an ensemble of twenty-nine structures in DPC at pH 6.0 gave a RMSD of 0.45 +/- 0.09 A for the region D168 to K182, while for all atoms it was 1.60 +/- 0.17 A. In SDS, the ensemble of nineteen structures each at pH 6.6 and 3.7 gave RMSDs of 0.28 +/- 0.07 A and 0.35 +/- 0.10 A, respectively, for the region D168 to K182. RMSD for superposition of all atoms was 1.36 +/- 0.10 A and 1.38 +/- 0.21 A at the respective pH values. In all cases a highly defined class A amphipathic helical structure was found for the region R171 to K182. Since the same structure occurs in micelles with either negatively charged or zwitterionic head groups it strongly suggests a dominant role for hydrophobic interactions in stabilizing the complex. The Y166 aromatic ring is bent back upon the helix axis at the lower pH. NMR determination of pKa values for D168, E169, E179 and E183 in the presence of SDS or DPC indicated a micro-pH at the micellar surface approximately one pH unit higher than the normal residue pKa. SDS interactions with the peptide were examined by collecting 1H NOESY spectra in the presence of protiated SDS. Residues R171, R173, R177, as well as the aromatic ring of Y166, were shown by intermolecular NOE measurements to interact with SDS, hence a key interaction in stabilizing the complex appears to be between interfacial basic side-chains and SDS alkyl chains.

Sphingosylphosphorylcholine (SPC), a putative product of sphingomyelin N-deacylation, has been shown to exert potent mitogenic activity (Desai, N.N. and Spiegel, S. (1991) Biochem. Biophys. Res. Commun. 181, 361-366). In order to explore potential mechanisms of action of SPC, the effects of SPC on endogenous protein phosphorylation was examined in vitro. In cytosol derived from Jurkat T cells, SPC was found to exert dual effects on protein phosphorylation. SPC (10-100 microM) induced the phosphorylation of a number of protein substrates with molecular weights of 32, 35, and 87 kDa. Higher concentrations of SPC (50-200 microM) inhibited the phosphorylation of proteins with estimated molecular weights of 22, 56, and 60 kDa by inhibiting the activity of endogenous protein kinases. Stimulation of protein kinases by SPC required distinct structural features (amino base and the hydrophobic character) from those required for inhibition of protein kinases (the choline phosphate headgroup as well as the hydrophobic character). The SPC-dependent protein kinases were distinct from protein kinase C, cyclic-nucleotide-dependent protein kinases, and calcium-dependent protein kinases, but may be related to casein kinase II. These studies suggest that SPC may act, at least in part, by modulating the activity of endogenous protein kinases.

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A2 (De Haas, G.H., Dijkman, R., Ransac, S. and Verger, R. (1990) Biochim. Biophys. Acta 1046, 249-257). Their inhibitory power appeared to be strictly controlled by the stereoconfiguration around the chiral C-2 atom and effective inhibition of the enzyme was observed only when incorporated into a micellar substrate-water interface. In the present study various direct binding techniques were applied to investigate the interaction of the enzyme with pure micelles of the stereoisomeric forms of 2-tetradecyl-amino-hexanol-1-phosphocholine (R-C14-PN and S-C14-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass = 14 kDa) on calibrated Superdex columns running in micellar solutions of R-C14-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa. Under identical conditions, micellar solutions of S-C14-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position. Light scattering experiments, ultrasedimentation and time-resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between enzyme and R-C14-PN micelles. The ultimate complex was shown to consist of four protein and about ten inhibitor molecules. Using time-resolved fluorescence spectroscopy the interaction was studied between the active site of phospholipase A2 and R-C14-PN molecules, both incorporated in an inert lipid matrix.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.

Rat serum phosphorylcholine binding protein (PCBP), a normal component of rat serum, inhibits in vitro aggregation of rat, rabbit and human platelets by interacting with platelets. In the present study, we have demonstrated the calcium-dependent, specific and saturable binding of 125I-PCBP to rat, rabbit and human platelets. Scatchard analysis of the binding data reveal a class of specific high-affinity binding sites with Kd values of 45.2 +/- 14.9, 26.1 +/- 8.3 and 32.2 +/- 9.9 nM on rat, rabbit and human platelets, respectively. These platelets also expressed a high capacity for binding to 125I-PCBP. The binding of 125I-PCBP to platelets was calcium- and time-dependent, and could be inhibited by phosphorylcholine (IC50 = 5.6 microM). Occupation of these binding sites by PCBP may be responsible for inhibition of platelet aggregation.

The interaction which occurs between an anti-lipid antibody (TEPC 15) and two phospholipids, phosphatidylcholine and phosphatidylethanolamine, when they are arranged in a lipid monolayer system has been studied. It is shown that the antibody is stabilised under the influence of a high lateral pressure when it is mixed with a lipid monolayer and that the behaviour of the antibody depends upon the lipid used. Measurements of the surface pressure and surface potential parameters of the lipid monolayers indicate that the antibody interacts differently with phosphatidylcholine compared with phosphatidylethanolamine. The antibody also exhibits a different interaction when it is pretreated with phosphorylcholine prior to being spread with a phosphatidylcholine monolayer. The interaction of the antibody with phosphatidylcholine-cholesterol monolayers has also been studied.

The aggregation behavior of cobra venom (Naja naja naja) phospholipase A2 in the presence of lipids and Ca2+ was examined using ultracentrifugation and crosslinking techniques. Velocity sedimentation experiments were performed in sucrose gradients. The sedimentation coefficients of the cobra phospholipase A2 and various controls, including bovine serum albumin (BSA), malate dehydrogenase, carbonic anhydrase and pancreatic phospholipase A2, were calculated both in the presence and absence of ligands. The monomeric phospholipid, diheptanoylphosphatidylcholine, and the phospholipid analogue, dodecylphosphocholine (DPC), increased the sedimentation coefficient of the cobra phospholipase A2 from 2.2 S to 2.9 S, a value that is consistent with the formation of an enzyme dimer. The control proteins were unaffected by the presence of phospholipid, except for BSA, which apparently binds large amounts of DPC. Crosslinking experiments with glutaraldehyde showed that in the presence of diheptanoylphosphatidylcholine or DPC, the amount of crosslinked enzyme increased. Ca2+ had no effect on the aggregation state of the enzyme as measured by either technique. Both the ultracentrifugation data and crosslinking data are consistent with the hypothesis that the cobra venom phospholipase A2 exists as a dimer or higher-order aggregate in the presence of lipid substrate, although it is yet to be determined whether the functional subunit is a monomer, dimer or higher-order oligomer.

Rat serum phosphorylcholine-binding protein (PCBP), also referred to as rat C-reactive protein, is present in serum under normal physiological conditions. In the present study, PCBP is shown to inhibit, in a dose-dependent manner, ADP- and platelet-activating factor (PAF)-induced platelet aggregation, using either washed rat platelets or platelet-rich plasma from rats or humans. Rat platelets, as expected, were refractory to aggregation by PAF. However, when rabbit antiserum against PCBP was added to rat platelet-rich plasma, it resulted in aggregation of platelets by PAF. PCBP also inhibited the PAF-induced aggregation of human platelets in a dose-dependent manner. The inhibition of ADP-induced platelet aggregation by PCBP was reversed by adding phosphorylcholine. Our results suggest that PCBP present in rat serum could be the cause of the refractory response of rat plasma to PAF. It is possible that PCBP inhibits platelet aggregation by binding to the platelet surface phospholipids.