Phosphatidylinositol Liposomes Research Articles

In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen–deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

Preparation of L-Glutamine Loaded Liposomes for Drug Delivery to Erythrocytes

Sickle cell disease (SCD) is a mortal chronic disease caused by a point mutation in the ? chain gene in the hemoglobin A (HbA) molecule. Erythrocyte polymerization in SCD is mostly seen as a result of the decrease in the amount of ions and water in the cell, i.e. dehydration and deoxygenation of erythrocytes. Deoxygenated and dehydrated erythrocytes become susceptible to clustering, causing clogging of blood vessels which then leads to crisis. Therefore, development of a new treatment method that can effectively prevent deoxygenation of erythrocytes or reduce the oxidative stress of sickle erythrocytes is one of the important issues. The aim of this study is to obtain a new lipid-based drug delivery system that will further be used for decreasing the oxidative stress of sickle erythrocytes. For the purpose, in this study, L-Glutamine (L-Gln) loaded liposomal drug delivery system composed of L-?-Phosphatidylinositol (PI) is prepared. Also, effect of encapsulated amount of L-Gln in liposomes is investigated. Liposomes are prepared via thin-film rehydration method. Characterizations of liposomes are implemented with pH measurements, zeta potential and size measurements. Erythrocytes and liposomes are incubated at 37ºC for 1 and 3 hours. Interactions between the erythrocytes and liposomes are investigated via optical microscopy and hemolysis experiments. The size and zeta potential of unloaded PI liposomes are determined as 89.01 nm with a polydispersity index of 0.438 and -23.4 ± 1.5 mV, respectively. Sizes of L-Gln loaded liposomes are obtained as 126.7, 148.6 and 197.2 nm for 20 mM, 40 mM and 60 mM of L-Gln, respectively. From the optical microscopy images, it is determined that as incubation period of erythrocytes and liposomes are increased, more liposomes are interacted with erythrocytes. Also, as L-Gln amount is increased within the liposomes, it was observed that erythrocytes preserve their morphology. Overall, with this study, it can be concluded that L-Glutamine loaded liposomes can be used as a new drug delivery platform for erythrocytes. Moreover, the results of this study provide preliminary steps and promising results for design and development of a lipid-based drug carrier system to be used in the treatment of specific erythrocyte-based diseases such as sickle-cell disease.

Lipidic Nanoparticles Comprising Phosphatidylinositol Mitigate Immunogenicity and Improve Efficacy of Recombinant Human Acid Alpha-Glucosidase in a Murine Model of Pompe Disease

Enzyme replacement therapy with recombinant human acid α-glucosidase (rhGAA) is complicated by the formation of anti-rhGAA antibodies, a short circulating half-life, instability in the plasma, and limited uptake into target tissue. Previously, we have demonstrated that phosphatidylinositol (PI) containing liposomes can reduce the immunogenicity and extend plasma survival of factor VIII (FVIII) in a mouse model of hemophilia A. In this article, we investigate the ability of PI liposomes to be used as a delivery vehicle to overcome the issues that complicate therapy with rhGAA. In a murine model of Pompe disease, administration of PI-rhGAA mitigated the immunogenicity of rhGAA, resulting in a significantly lower formation of anti-rhGAA antibodies. PI-rhGAA also showed minimal improvements to the pharmacokinetic parameters and efficacy measures compared to free rhGAA. Overall, these data suggest that PI-rhGAA may have the potential to be a useful therapeutic option for improving the treatment of Pompe disease.

Phosphatidylinositol inhibits respiratory syncytial virus infection

Respiratory syncytial virus (RSV) infects nearly all children under age 2, and reinfection occurs throughout life, seriously impacting adults with chronic pulmonary diseases. Recent data demonstrate that the anionic pulmonary surfactant lipid phosphatidylglycerol (PG) exerts a potent antiviral effect against RSV in vitro and in vivo. Phosphatidylinositol (PI) is also an anionic pulmonary surfactant phospholipid, and we tested its antiviral activity. PI liposomes completely suppress interleukin-8 production from BEAS2B epithelial cells challenged with RSV. The presence of PI during viral challenge in vitro reduces infection by a factor of >10(3). PI binds RSV with high affinity, preventing virus attachment to epithelial cells. Intranasal inoculation with PI along with RSV in mice reduces the viral burden 30-fold, eliminates the influx of inflammatory cells, and reduces tissue histopathology. Pharmacological doses of PI persist for >6 h in mouse lung. Pretreatment of mice with PI at 2 h prior to viral infection effectively suppresses inflammation and reduces the viral burden by 85%. These data demonstrate that PI has potent antiviral properties, a long residence time in the extracellular bronchoalveolar compartment, and a significant prophylaxis window. The findings demonstrate PG and PI have complementary roles as intrinsic, innate immune antiviral mediators in the lung.

Critical Role of Arg/Lys343 in the Species-Dependent Recognition of Phosphatidylinositol by Pulmonary Surfactant Protein D,

Surfactant protein D (SP-D) plays important roles in lung host defense. However, it can also recognize specific host molecules and contributes to surfactant homeostasis. The major known surfactant-associated ligand is phosphatidylinositol (PI). Trimeric neck-carbohydrate recognition domains (NCRDs) of rat and human SP-D exhibited dose-dependent, calcium-dependent, and inositol-sensitive binding to solid-phase PI and to multilamellar PI liposomes. However, the rat protein exhibited a >5-fold higher affinity for solid-phase PI than the human NCRD. In addition, human dodecamers, but not full-length human trimers, efficiently coprecipitated with multilamellar PI liposomes in the presence of calcium. A human NCRD mutant resembling the rat and mouse proteins at position 343 (hR343K) showed much stronger binding to PI. A reciprocal rat mutant with arginine at the position of lysine 343 (rK343R) showed weak binding to PI, even weaker than that of the wild-type human protein. Crystal complexes of the human trimeric NCRD with myoinositol and inositol 1-phosphate showed binding of the equatorial OH groups of the cyclitol ring of the inositol to calcium at the carbohydrate binding site. Myoinositol binding occurred in two major orientations, while inositol 1-phosphate appeared primarily constrained to a single, different orientation. Our studies directly implicate the CRD in PI binding and reveal unexpected species differences in PI recognition that can be largely attributed to the side chain of residue 343. In addition, the studies indicate that oligomerization of trimeric subunits is an important determinant of recognition of PI by human SP-D.

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5–1000 μM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water–lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10–1000 μM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Activation of dendritic cells by liposomes prepared from phosphatidylinositol mannosides from Mycobacterium bovis bacillus Calmette-Guerin and adjuvant activity in vivo.

Liposome vesicles could be formed at 65 degrees C from the chloroform-soluble, total polar lipids (TPL) extracted from Mycobacterium bovis bacillus Calmette-Guérin (BCG). Mice immunized with ovalbumin (OVA) entrapped in TPL liposomes produced both anti-OVA antibody and cytotoxic T lymphocyte responses. Murine bone marrow-derived dendritic cells were activated to secrete interleukin-6 (IL-6), IL-12, and tumor necrosis factor upon exposure to antigen-free TPL liposomes. Three phosphoglycolipids and three phospholipids comprising 96% of TPL were identified as phosphatidylinositol dimannoside, palmitoyl-phosphatidylinositol dimannoside, dipalmitoyl-phosphatidylinositol dimannoside, phosphatidylinositol, phosphatidylethanolamine, and cardiolipin. The activation of dendritic cells by liposomes prepared from each purified lipid component of TPL was evaluated in vitro. A basal activity of phosphatidylinositol liposomes to activate proinflammatory cytokine production appeared to be attributable to the tuberculosteric fatty acyl 19:0 chain characteristic of mycobacterial glycerolipids, as similar lipids lacking tuberculosteric chains showed little activity. Phosphatidylinositol dimannoside was identified as the primary lipid that activated dendritic cells to produce amounts of proinflammatory cytokines several times higher than the basal level, indicating the importance of mannose residues. Although the activity of phosphatidylinositol dimannoside was little influenced by palmitoylation of mannose at C-6, a further palmitoylation at inositol C-3 diminished the induction levels of IL-6 and IL-12. Further, OVA entrapped in palmitoyl-phosphatidylinositol dimannoside liposomes was delivered to dendritic cells for major histocompatibility complex class I presentation more effectively than TPL OVA-liposomes. BCG liposomes containing mannose lipids caused up-regulation of costimulatory molecules and CD40. Thus, the inclusion of pure phosphatidylinositol mannosides of BCG in lipid vesicle vaccines represents a simple and efficient option for targeting antigen delivery and providing immune stimulation.

A common pathway for the uptake of surfactant lipids by alveolar cells.

The uptake of different surfactant lipids-dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), or phosphatidylinositol (PI)-and liposomes with a surfactant-like composition by alveolar type II cells (alveolar type II cells) and macrophages (alveolar macrophages) was studied in vitro. Fluorescent-labeled liposomes containing either 86% of the studied lipid, i.e., DPPC, PG, PI, and 6% labeled phosphatidylethanolamine (PE) and 8% cholesterol or a lipid mixture similar to surfactant (DPPC, PG, PI, phosphatidylcholine, PE, and cholesterol in a weight ratio of 55:8:2:21:8:6) were incubated with alveolar macrophages and alveolar type II cells. The cell-associated fluorescence assessed by flow cytometry demonstrated a higher uptake of PG and PI by both alveolar macrophages and alveolar type II cells, and a lower uptake of DPPC by alveolar macrophages. In addition, fewer alveolar type II cells take up DPPC, whereas there are no differences for the alveolar macrophages in the number of cells involved in the uptake. Competition experiments with Texas Red-labeled liposomes and either DPPC liposomes or PI liposomes labeled with Bodipy indicated that all these liposomes are internalized via the same pathway by alveolar cells. Thus, lipid composition directly influences the (re)uptake of surfactant.

Affinity of various bio-trace elements to lipid membrane: In vivo and in vitro study using multitracer

Affinity of several bio-trace elements to various reconstructed lipid membranes (liposomes) was assessed using multitracer analysis technique (MTAT). To estimate affinity of bio-trace elements to the sub-cellular components such as lipid membranes, MTAT was applied to the various reconstructed liposomes. Phosphatidylcholine and sphingomyelin liposomes showed a similar pattern of elemental affinity. Phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol liposomes showed another similar pattern of elemental affinity, except that phosphatidylinositol liposome has no affinity to vanadium.

Differential interaction of Sophora isoflavonoids with lipid bilayers

The mechanisms of some biological effects exerted by flavonoids (e.g. activity against lipid oxidation, multidrug resistance modulation) may involve their interactions with lipid bilayers. Due to variety of substituents attached to the flavonoid nucleus individual isoflavones significantly differ in their properties; in particular they may differently interact with membranes. For this reason we have investigated the interactions of different isoflavones with lipid bilayers. The influence of four plant isoflavones on the phase transitions of dipalmitoylphosphatidylcholine (DPPC) and on liposome aggregation was studied, using microcalorimetry and absorption measurements, respectively. We found that isoflavones substituted with one or two prenyl groups less effectively induce liposome aggregation than more polar ones, possessing no prenyl groups. For aggregation-promoting compounds, rather small differences in the influence on phosphatidylcholine, phosphatidylserine and phosphatidylinositol liposomes were recorded. On the other hand, the alteration of DPPC phase transitions by prenyl-substituted isoflavones was more pronounced than changes induced by non-prenyl ones. On the basis of observed effects we conclude that prenyl-substituted isoflavones penetrate deeper into the lipid bilayer while more polar ones act closer to the membrane surface. Comparing our results with biological tests it seems that interactions with the hydrophobic core of membranes are responsible for the activity of the studied isoflavones.

The effect of grafted poly(ethylene glycol) on the electrophoretic properties of phospholipid liposomes and their adsorption to bacterial biofilms

Liposomes have been prepared from the phospholipids, dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholine (DMPC, DPPC, DSPC, respectively) containing dimethyldioctadecylammonium bromide (DDAB) or phosphatidylinositol (PI) and variable amounts (0–9 mole%) of dipalmitoylphosphatidylethanolamine bonded to poly(ethylene glycol) [PEG] of molecular mass 2000 (DPPE-PEG-2000). The electrophoretic mobilities of the liposomes and corresponding zeta potentials were found to decrease in magnitude for both cationic (DDAB) and anionic (PI) liposomes with increasing DPPE-PEG-2000 incorporation. The chain length of the major lipids in the liposomes (DMPC, DPPC and DSPC) did not significantly affect the zeta potentials of cationic liposomes but had a small effect on anionic liposomes. The zeta potentials were larger for DMPC–PI liposomes where the acyl chains were in the lamellar liquid crystalline phase. The adsorption of the liposomes to immobilised biofilms of Staphylococcus aureus was found to be represented satisfactorily by the Langmuir isotherm. The affinity of the liposomes for the biofilms was inhibited by pegylation suggesting that the ‘stealth’ property of pegylated liposomes applies to bacterial biofilms. Both the surface coverage and the magnitude of the Gibbs energy of adsorption decrease with the extent of pegylation.

RPE65, the Major Retinal Pigment Epithelium Microsomal Membrane Protein, Associates with Phospholipid Liposomes

The retinal pigment epithelium (RPE)-specific protein RPE65 is the major protein of the RPE microsomal membrane fraction. Though RPE65 lacks transmembrane domains or signal peptide, detergents are required for its maximally effective solubilization in isotonic buffers. However, in 0.75–1.0mKCl, RPE65 is as soluble without detergent, indicating a peripheral membrane association. We wished to understand why this non-membrane-inserted protein was so closely associated with RPE microsomal membranes. To explore the possible involvement of interactions with phospholipids, an isotonic salt-soluble extract of RPE was incubated with phosphatidylcholine (PC)/phosphatidylserine (PS)/phosphatidylinositol liposomes and centrifuged to sediment the liposomes. RPE65 cosedimented with the liposome pellet. RPE65 also cosedimented with synthetic dipalmitoyl-, 1-palmitoyl, 2-docosahexaenoyl-PC or dipalmitoyl-PS liposomes. Incubation with 1 mmCa2+or 1 mmEGTA had no effect, indicating a Ca2+-independent association. A spectrophotometric assay showed that this interaction of RPE65 with phospholipid vesicles resulted in increased light scattering, consistent with phospholipid vesicle aggregation. Resonance energy transfer experiments showed that any putative aggregation occurred without subsequent vesicle fusion. This PC affinity was further confirmed by incubation of RPE extract with dimyristoyl-PC-immobilized artificial membrane (IAM.PC) matrix. The RPE65 selectively bound and was elutable with 2% detergent. This RPE65–phospholipid liposome association may explain the solubilization characteristics of RPE65 and may be related to the function of RPE65 and to its physical association with the RPE smooth endoplasmic reticulum.

Binding of Anionic Phospholipids to Retinal Pigment Epithelium May Be Mediated by the Scavenger Receptor CD36

The specific recognition of negatively charged phospholipids in cell membranes has been suggested to play an important role in a variety of physiological and pathophysiological processes. Recent work (Rigotti, A., Acton, S. L., and Krieger, M. (1995) J. Biol. Chem. 270, 16221-16224) has described specific and tight binding of anionic phospholipids, such as phosphatidylserine (PS) and phosphatidylinositol (PI), to the class B scavenger receptors, CD36 and SR-B1. We have previously reported that CD36 is present on retinal pigment epithelium (RPE) and plays a role in the phagocytosis of photoreceptor outer segments (ROS), a function critical to the normal visual process (Ryeom, S. W., Sparrow, J. R., and Silverstein, R. L. (1996) J. Cell Sci. 109, 387-395). We now report that phospholipid liposomes PS and PI, but not phosphatidylethanolamine, bind specifically to RPE. Cross-competition experiments suggest that PS and PI recognize the same receptor on RPE, while immunoinhibition studies indicate that the receptor is CD36. RPE cells isolated from a mutant rat strain, the RPE of which does not express CD36 ( Sparrow, J. R., Ryeom, S. W. , Abumrad, N., Ibrahimi, A., and Silverstein, R. L. (1996) Exp. Eye Res., in press), did not bind PS or PI, further confirming the role of CD36. We also showed that purified ROS blocked binding and uptake of anionic phospholipid liposomes by RPE and that PS and PI liposomes blocked ROS uptake by RPE, suggesting that PS and PI on the ROS membrane may be the ligands on ROS recognized by CD36. This is the first demonstration that CD36-phospholipid interactions may play a role in normal physiology.

Altered Carbohydrate Recognition Specificity Engineered into Surfactant Protein D Reveals Different Binding Mechanisms for Phosphatidylinositol and Glucosylceramide

Pulmonary surfactant protein D (SP-D) is a member of the collection subgroup of the C-type lectin superfamily that binds glycosylated lipids such as phosphatidylinositol (PI) and glucosylceramide (GlcCer). We have previously reported that the carbohydrate recognition domain of SP-D plays an essential role in lipid binding. However, it is unclear how the carbohydrate binding property of SP-D contributes to the lipid binding. To clarify the relationship between the lectin property and the lipid binding activity of rat SP-D, we expressed wild-type recombinant rat SP-D (rSP-D) and a mutant form of the protein with substitutions Glu-321-->Gln and Asn-323-->Asp (SP-DE321Q,N323D) in CHO-K1 cells. The indicated mutations have previously been shown to change the carbohydrate binding specificity of surfactant protein A and mannose-binding protein from mannose > galactose to the converse. rSP-D expressed in mammalian cells was essentially identical to native rat SP-D in its lipid and carbohydrate binding properties. In contrast, SP-DE321Q,N323D was unable to bind GlcCer, but retained binding activity toward PI liposomes and solid-phase PI. The efficiency of SP-DE321Q,N323D binding to PI liposome was approximately 50% of that of rSP-D in the presence of 5 mM Ca2+, but equivalent at 20 mM Ca2+. Carbohydrates competed for SP-D binding to PI such that maltose > galactose for rSP-D, and the order was reversed for SP-DE321Q,N323D. Furthermore, SP-DE321Q,N323D could bind to digalactosyldiacylglycerol more effectively than rSP-D. These results suggest the following. 1) The carbohydrate binding specificity of SP-DE321Q,N323D was changed from a mannose-glucose type to a galactose type; 2) the GlcCer binding property of SP-D is closely related to its sugar binding activity; and 3) the PI binding activity is not completely dependent on its carbohydrate binding specificity.

Chimeras of surfactant proteins A and D identify the carbohydrate recognition domains as essential for phospholipid interaction.

Pulmonary surfactant proteins A (SP-A) and D (SP-D) possess similar structure as members of the mammalian C-type lectin superfamily. Both proteins are composed of four characteristic domains which are: 1) an NH2-terminal domain involved in interchain disulfide formation (denoted A1 domain for SP-A or D1 for SP-D); 2) a collagenous domain (denoted A2 or D2); 3) a neck domain (denoted A3 or D3); and 4) a carbohydrate recognition domain (denoted A4 or D4). SP-A specifically binds to dipalmitoylphosphatidylcholine, the major lipid component of surfactant, and can regulate the secretion and recycling of this lipid by alveolar type II cells. SP-D binds to phosphatidylinositol (PI) and glucosylceramide (GlcCer), and its role in alveolar lipid metabolism remains to be clarified. To understand the relationship between the structure and the function of both proteins with respect to their interaction with lipids, we expressed recombinant wild type rat SP-D (rSP-D) and chimeric molecules of SP-A and SP-D (A1A2A3D4, A1A2D3D4, and D1D2A3A4) using a baculovirus expression system, and performed lipid binding and aggregation assays. The rSP-D effectively competed with 125I-labeled native rat SP-D in a solid phase binding assay to PI and GlcCer in a manner nearly identical to native SP-D. The rSP-D also bound to PI liposomes with approximately half the affinity of native rat SP-D. Chimera A1A2D3D4 competed with iodinated SP-D in the solid phase binding assay to both PI and GlcCer. This chimera did not bind to dipalmitoylphosphatidylcholine (DPPC) liposomes or induce their aggregation. Chimera A1A2A3D4 did not bind solid phase PI or GlcCer but was equivalent to rSP-D in binding to PI liposomes. This chimera exhibited weak binding to DPPC but failed to aggregate DPPC liposomes. Chimera D1D2A3A4 failed to bind PI and GlcCer and bound weakly to DPPC liposomes but was quite effective at inducing aggregation of DPPC liposomes. These findings demonstrate that the D3 plus D4 domains of SP-D play a role in lipid binding and that the D4 domain is essential for PI binding. Furthermore, the A3 domain of SP-A cannot account for all the lipid binding activity of this protein. In addition, the results implicate the A4 domain of SP-A as an important structural domain in lipid aggregation phenomena.

Inhibitory immunoglobulin M antibodies to tumor necrosis factor-inducing toxins in patients with malaria

Cytokines such as tumor necrosis factor alpha (TNF) appear to play an important role in the pathogenesis of malaria. We have previously shown that TNF is produced in response to substances released at schizont rupture, which we have called malaria toxins. In mice these toxins stimulate a T cell-independent antibody response, generating short-lived immunoglobulin M (IgM) antibodies that inhibit the TNF-inducing activity of the toxins. We report here that a similar antibody response is seen in humans. Serum from a European adult infected with Plasmodium falciparum inhibited the induction of TNF by malaria toxins derived from P. falciparum-infected erythrocytes. We found that IgM antibodies were responsible for the inhibitory activity. These inhibitory antibodies could not be detected in convalescent-phase serum collected from the same patient 6 weeks later or in sera from healthy European and African controls. The antibodies appeared to be malaria specific in that they inhibited TNF induction by a variety of P. falciparum isolates but failed to inhibit TNF induction by bacterial lipopolysaccharide or lipoteichoic acid. The inhibitory antibodies bound to liposomes containing phosphatidylinositol but not other phospholipids. Serum from a European adult infected with P. vivax also inhibited the activity of toxins derived from P. falciparum-infected erythrocytes, and this too was mediated by IgM antibodies which were malaria specific and bound to phosphatidylinositol liposomes.

Effect of extracellular phosphatidylinositol on c- myc gene-expressed human renal cancer cell line

Effect of exogenously added soybean phosphatidylinositol on c- myc gene expressed and unexpressed human cancer cell lines was investigated. When phosphatidylinositol liposomes were introduced into culture media, viability of c- myc unexpressed cells was reduced, while that of c- myc expressed cells was not. Death of c- myc unexpressed cells by phosphatidylinositol liposomes was found to be caused by abnormally accumulated intracellular Ca 2+, and it seemed to be related to reduction of protein kinase C activity.

Modulation of tissue plasminogen activator biosynthesis by phosphatidylinositol liposomes in human fetal lung fibroblasts

Phosphatidylinositol (PI) liposomes at 40 microM increased tissue plasminogen activator (t-PA) biosynthesis by human fetal lung fibroblasts IMR-90 (FLF), after 5 days of incubation by 7.4 +/- 1.4 times of the control level. Other phospholipid liposomes, such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylglycerol (PG), had no effect on t-PA biosynthesis by FLF. The induction of t-PA biosynthesis by PI liposomes was inhibited by specific inhibitors of phosphoinositide pathway: gentamycin and lithium chloride. Thus, gentamycin inhibited the effect of PI liposomes on t-PA biosynthesis by 76% (P less than 0.001), while it had no effect on control FLF. Likewise, lithium chloride inhibited t-PA biosynthesis of both PI-treated and control FLF by greater than 84%. The induction of t-PA biosynthesis by PI liposomes was dependent on RNA transcription and independent of DNA biosynthesis.

Phosphatidylinositol liposomes increase calcium uptake and tissue plasminogen activator secretion by fetal human lung fibroblasts

PtdIns liposomes, at a concentration of 40 microM, induced in FLF the synthesis of t-PA-Ag, and enhanced 45Ca2+ uptake. The induction of t-PA-Ag biosynthesis by PtdIns liposomes in FLF was inhibited by 5-15 microM verapamil, an inhibitor of Ca2+ uptake via the so-called "slow channels" by 0.5-10 microM TFP, an inhibitor of Ca2+ transport ATPase, and by 10-90 microM TMB-8, an inhibitor of intracellular Ca2+ mobilization. t-PA-Ag secretion was inhibited by decreasing the Ca2+ concentration less than 1.2 mM. On the other hand, addition of 0.08 microM of calcium ionophore A23187 increased t-PA-Ag biosynthesis after 72 hr of incubation by 247% (P less than 0.01). These data support previous results and indicate that the synthesis of t-PA in FLF is Ca2+ dependent. Thus, it is suggested that PtdIns liposomes increase t-PA biosynthesis by affecting calcium metabolism.