Outer Leaflet Of Plasma Membrane Research Articles

Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers

Primary deficiency of merosin causes a severe congenital muscular dystrophy (CMD) and a mouse dystrophy (dy/dy mouse). Also, its secondary deficiency is seen in some CMD with abnormal glycosylation of Alpha-dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via Beta-dystroglycan, a transmembrane protein. In immunogold and freeze-etch electron microscopic studies, merosin in basal lamina of normal skeletal muscles has a zonation in the distribution and is localized at the lamina lucida of muscle basal lamina, and dystrophin molecules are often closed to merosin molecules at the inside and outside surface of muscle plasma membrane. Moreover, merosin molecules exist as the short fine cross-bridge fibrils connecting the basal lamina to the neighboring outer leaflet of the muscle plasma membrane. In freeze-fracture studies, the changes in muscle plasma membranes of dy/dy mice reveal a markedly decreased density of orthogonal arrays (OAs) but normal density of intramembranous particles (IMPs), whereas depletions of IMPs with decreased OAs have been found in Fukyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, and mdx mice. Thus, further studies including the functional role of OAs would be required to understand the pathomechanism of merosin-deficient CMD.

New phospholipase A(2) isozymes with a potential role in atherosclerosis.

Inflammation is an integral feature of atherosclerosis, in which inflammatory processes contribute to the initiation, progression and rupture of lipid-rich atherosclerotic plaques. Recent studies have suggested the involvement of the proinflammatory secretory phospholipase A2 (sPLA2)-IIA in the development of atherosclerosis. This enzyme has been proposed to hydrolyze phosphatidylcholine (PC) in lipoproteins to liberate lyso-PC and free fatty acids in the arterial wall, thereby facilitating the accumulation of bioactive lipids and modified lipoproteins in atherosclerotic foci. However, the recent discovery of several novel sPLA2 isozymes has raised the question of which types of sPLA2 truly contribute to the atherosclerotic process. Amongst the 10 mammalian sPLA2 isozymes, sPLA2-X, -V, -IIF and -III exhibit much more potent PC-hydrolyzing activity than do the others, and can release free fatty acids and lysophospholipids from the PC-rich outer leaflet of the cellular plasma membrane. In particular, sPLA2-X and sPLA2-V hydrolyze PC in lipoproteins far more efficiently than does sPLA2-IIA. Moreover, sPLA2-X promotes foam cell formation in vitro and is expressed in the atherosclerotic arterial walls of apolipoprotein E deficient mice in vivo. PC-hydrolyzing sPLA2 isozymes, particularly sPLA2-V and sPLA2-X, are attractive candidates for proatherosclerotic factors that may act in place of sPLA2-IIA. However, their expression in human atherosclerotic lesions requires confirmation by specific methods that can distinguish between the different sPLA2 isozymes.

Mechanical constraint imposed on plasma membrane through transverse phospholipid imbalance induces reversible actin polymerization via phosphoinositide 3-kinase activation.

Platelets were used to explore the effect of membrane curvature induced by phospholipid excess on cell shape and on organization of the actin cytoskeleton. We showed that the addition of short chain analogues of phospholipids to the outer leaflet of plasma membrane of resting platelets immediately induced a shape change with long filopodia formation containing newly polymerized actin. Cells recovered rapidly their discoid shape and their initial F-actin content only with the phosphatidylserine analogue, which was transported to the inner leaflet by aminophospholipid translocase. Filopodia formation and actin polymerization were inhibited in platelets pre-incubated with cytochalasin D. Both wortmannin and LY294002, two unrelated inhibitors of phosphoinositide 3-kinase, considerably reduced actin polymerization and filopodia formation. Phospholipid imbalance was accompanied by a reversible translocation of phosphoinositide 3-kinase from cytoplasm to plasma membrane. In agreement with a role for PI 3-kinase, when phospholipids were added to platelets, PtdIns(3,4)P2 increased two-fold and Akt protein was partly phosphorylated. A similar shape change was also observed in nocodazole-treated L929 fibroblasts which were incubated with the similar phospholipid analogues. In those nucleated cells, where the microtubule cytoskeleton was disrupted, a major actin-dependent membrane extension was induced by addition of short chain phospholipids that required the functional integrity of PI 3-kinase. We conclude that any physical constraint acting on plasma membrane and resulting on local changes in membrane curvature is sufficient to initiate transient actin polymerization via phosphoinositide 3-kinase activation.

Annexin I Is an Endogenous Ligand that Mediates Apoptotic Cell Engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Group B streptococcus (GBS) modifies macrophage phosphatidylserine metabolism during induction of apoptosis

Group B streptococcus (GBS) induced macrophage apoptosis by which it could avoid host defence mechanisms. Macrophages, which constitutively express phosphatidylserine (PtdSer) on the outer leaflet of plasma membrane, increased PtdSer exposure during GBS-induced apoptosis. Induction of apoptosis decreased PtdSer radioactivity of macrophages incubated with [ 3H]serine. The effect appeared not due to increasing conversion of PtdSer to phosphatidylethanolamine or phosphatidylcholine nor to the release of radioactive membrane vesicles. The radioactivity in lysoPtdSer was also reduced. These results confirm that induction of apoptosis involves a modification of PtdSer metabolism and point out the typical features of the GBS-induced apoptosis with respect to other models of apoptosis.

Effect of different incubation conditions on phosphatidylserine externalization and motion parameters of purified fractions of highly motile human spermatozoa.

The objective of this study was to assess the temporal effects of sperm incubation at body temperature with various amounts of human serum albumin (HSA) on motion parameters and phosphatidylserine externalization, an expression of membrane integrity. Purified sperm populations were prepared by discontinuous gradient separation, incubated at 37 degrees C in 3 different culture conditions (human tubal fluid [HTF] alone, HTF plus 0.3% HSA, and HTF plus 3% HSA) and evaluated at 0, 1, 3, 6, and 24 hours. Annexin V binding was used to monitor membrane translocation of phosphatidylserine and a computer-assisted semen analyzer was used to evaluate motion parameters. All incubation conditions led to a time-dependent, significant decline in sperm motion parameters and an increase in exposure of phosphatidylserine (annexin V+, live cells) to the outer leaflet of the plasma membrane in both patients and donors. Patients had a higher degree of motility loss and externalization of phosphatidylserine than donors. The decline in the percentage of normal cells (annexin V-, live) was greater in HTF alone up to 6 hours, and the decline in the percentages of motile and rapid sperm were greater in HTF alone throughout 24 hours when compared with HSA supplementation. We conclude that prolonged incubation of purified populations of highly motile human spermatozoa at body temperature was associated with significant motility loss and membrane changes as revealed by phosphatidylserine translocation. A higher concentration of HSA resulted in a relative protective effect against such impairments, particularly during the first 6 hours of incubation. Under the experimental conditions tested, significant differences were observed between infertile men and fertile controls.

Frequent reversible membrane damage in peripheral blood B cells in human T cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP).

Apoptosis in peripheral blood lymphocyte populations in HTLV-I-infected people in vivo was examined, to study the lymphocyte dynamics in HTLV-I infection. Freshly isolated lymphocytes from 10 non-infected healthy people, eight asymptomatic HTLV-I carriers and 15 patients with HAM/TSP were stained with FITC-labelled annexin V to detect phosphatidylserine (PS) residue exposure at the outer plasma membrane leaflet as an early marker of apoptosis. There was no significant difference in annexin V positivity in CD4+ and CD8+ lymphocytes between non-infected subjects, asymptomatic carriers and HAM/TSP patients, but there was a greatly increased exposure of PS on CD19+ lymphocytes (B cells) detected by FITC-annexin V in 12 out of 15 (80%) HAM/TSP patients, while only two out of eight (25%) asymptomatic carriers and none of the non-infected healthy people showed this aberrant PS exposure on B cells. The intensity of annexin V staining of B cells in HAM/TSP was intermediate, as distinct from the high annexin V staining on advanced apoptotic cells. However, annexin V positivity was decreased when the cells were stained after 24 h of culture, suggesting that the intermediate PS exposure on the B cell in HAM/TSP is not a consequence of an apoptotic process, but rather reflects reversible membrane damage. B cells with PS exposure in vivo might provide a site for coagulation and inflammation, and so contribute to the pathogenesis of HAM/TSP and its complications.

Modulation of the Susceptibility of Human Erythrocytes to Snake Venom Myotoxic Phospholipases A2: Role of Negatively Charged Phospholipids as Potential Membrane Binding Sites

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp-49 and Lys-49 phospholipases A2 (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.

Domain Formation in Models of the Renal Brush Border Membrane Outer Leaflet

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20–50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.

3P238細胞膜外葉におけるフォスファチジルセリン分布の時間変化の可視化

3P238細胞膜外葉におけるフォスファチジルセリン分布の時間変化の可視化

Molecular composition and function of the slit diaphragm: nephrin, the molecule responsible for proteinuria

Although the role of the glomerular basement membrane has been emphasized as the barrier for retaining plasma proteins, some recent studies have clearly shown that the slit diaphragm is the structure most likely to be the main barrier in the glomerular capillary wall. Nephrin, ZO-1, P-cadherin and FAT are reported to be components of the slit diaphragm. Of these molecules, the molecular natures of ZO-1 and nephrin are relatively well investigated. ZO-1 was originally identified as a component of the tight junction. Nephrin was identified as a gene product mutated in congenital nephrotic syndrome. ZO-1 is localized at the cytoplasmic surface just at the insertion point of the slit diaphragm, whereas nephrin is located at the outer leaflet of plasma membranes. The expression of nephrin is decreased in several kinds of proteinuric states, and anti-nephrin monoclonal antibody (mAb) 5-1-6 is capable of inducing massive proteinuria, which indicates that nephrin is a functional protein in the slit diaphragm. Nephrin is expected to be a target of therapy for proteinuria. A model of nephrin assembly that forms the structure of the slit diaphragm has been proposed. However, we observed that the slit diaphragm structure was maintained in mAb 5-1-6 nephropathy, although nephrin expression was dramatically decreased. These findings suggest that nephrin is not essential for maintaining the structural integrity of the slit diaphragm. Although it has been proposed that P-cadherin is a core protein in the slit diaphragm, this hypothesis is still controversial. To better understand the structure of the slit diaphragm, it is particularly important to identify other components that build up the structure of the slit diaphragm together with nephrin and ZO-1.

Stimulated Nonspecific Transport of Phospholipids Results in Elevated External Appearance of Phosphatidylserine in ras-Transformed Fibroblasts

The content of phosphatidylserine (PS) was found to be increased three times in the plasma membrane outer leaflet of ras-transformed fibroblasts compared to their nontransformed counterparts. In an attempt to determine the mechanisms responsible for the enhanced external appearance of PS, we investigated the activities of aminophospholipid translocase and the nonspecific lipid scramblase. Both transport systems could separately or in combination contribute to PS accumulation in the extracellular leaflet. Aminophospholipid transfer was assessed by measuring the rate of NBD-PS internalization, and scramblase activity was estimated from the internalization of NBD-PC. The results showed that the aminophospholipid transport was inhibited and the nonspecific transport was stimulated in ras-transformed cells. To assess which of these two transport systems was related to elevation of PS external appearance, each of them was submitted to reversible alterations and the content of PS was measured simultaneously. Aminophospholipid translocase activity was inhibited by pyridyldithioethylamine treatment and reversed by reduction with dithiothreitol. Scramblase activity was modulated by a calcium repletion–depletion procedure. Calcium depletion was performed by cell incubation with BAPTA-AM and EGTA as Ca2+ intracellular and extracellular chelators. Restoration of the intracellular Ca2+ was achieved by cell incubation with Ca2+ and Ca2+-ionophore A23187. The results showed that the changes in PS outer appearance did not correlate with the uptake of NBD-PS but were closely related to NBD-PC internalization, suggesting that the nonspecific bidirectional lipid transfer was the major transport system translocating PS to the outer leaflet in ras-transformed cells.

Regulation of Phospholipid Scramblase Activity during Apoptosis and Cell Activation by Protein Kinase Cδ

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.

Regulation of phosphatidylserine exposure at the cell surface by the serine–base exchange enzyme system during CD95-induced apoptosis

Early in the apoptotic process, CD95 induces a translocation of phosphatidylserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium-dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells. We found that CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompanied both by a decreased PtdSer synthesis and by a strong reduction of CD95-induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V–fluorescein isothiocyanate (FITC)-labeled cells. PtdSer synthesis through the serine–base exchange enzyme system thus appeared as one of the mechanisms implicated in the recently discovered CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversely, increasing the activity of the serine–base exchange enzyme system with different drugs, either the K + channel blocker quinine, the cationic amphiphil stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), resulted in an increased appearance of PtdSer at the surface of CD95-treated cells. Both PtdSer synthesis and CD95-induced annexin V–FITC reactivity were abrogated in ATP-depleted cells. Also, modifying the membrane potential with valinomycin (hyperpolarization) or either gramicidin or KCl (depolarization) demonstrated a strong relationship between PtdSer synthesis and annexin V–FITC reactivity in CD95-treated cells. Together, our results indicate that CD95-induced exposure of PtdSer at the cell surface could be regulated by the activity of the serine–base exchange enzyme system.

51] Estimating sphingolipid metabolism and trafficking in cultured cells using radiolabeled compounds

Publisher Summary The use of radioactive metabolites, particularly radiolabeled complex sphingolipids, represents an experimental approach to investigating different aspects of sphingolipid metabolism. The rationale of this approach is based on the fact that when complex sphingolipids—sphingomyelin and gangliosides—are added to cultured cells, they are partly taken up and inserted into the outer plasma membrane leaflet (where most cell sphingolipids are located); they are then processed metabolically through the intracellular traffic and metabolizing pathways of the corresponding endogenous components. Assessing the metabolic products formed by cells from an exogenously administered sphingolipid can be instrumental in (1) recognizing the metabolic processing and intracellular trafficking of a specific sphingolipid, (2) ascertaining cell specificity in metabolic processes, and (3) establishing the subcellular aspects of metabolic events involved in sphingolipid processing. It can also be useful in investigating the role of sphingolipids in signaling processes and evaluating the effects exerted by different molecules on sphingolipid metabolism. All this information can be gained using appropriately radiolabeled complex sphingolipids, adequate pulse-chase conditions, and carefully separating, identifying, and quantifying the radiolabeled metabolites. This chapter describes the experimental procedures useful for a global investigation of radiolabeled sphingolipid metabolism in cultured cells and then the specific protocols suitable for studying specific pathways involved in sphingolipid metabolism and intracellular sphingolipid trafficking.

Polyamine Regulation of Plasma Membrane Phospholipid Flip-Flop during Apoptosis

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.

Ca2+-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions

In multicellular organisms, various compositionally distinct fluid compartments are established by epithelial and endothelial cellular sheets. For these cells to function as barriers, tight junctions (TJs) are considered to create a primary barrier for the diffusion of solutes through the paracellular pathway [–3]. In ultrathin sections viewed under electron microscopy, TJs appear as a series of apparent fusions, involving the outer leaflets of plasma membranes of adjacent cells, to form the so-called kissing points of TJs, where the intercellular space is completely obliterated [4]. Claudins are a family of 16 proteins whose members have been identified as major integral membrane proteins localized exclusively at TJs [5–8]. It remains unclear, however, whether claudins have the cell-adhesion activity that would explain the unusual intercellular adhesion at TJs. Using mouse L-fibroblast transfectants expressing various amounts of claudin-1, -2 or -3, we found that these claudins possess Ca2+-independent cell-adhesion activity. Using ultrathin-section electron microscopy, we observed many kissing points of TJs between adjacent transfectants. Furthermore, the cell-adhesion activity of occludin, another integral membrane protein localized at TJs [9–11], was negligible when compared with that of claudins. Thus, claudins are responsible for TJ-specific obliteration of the intercellular space.

Effects of Docosahexaenoic and Arachidonic Acids on the Synthesis and Distribution of Aminophospholipids during Neuronal Differentiation of PC12 Cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.

REVERSIBLE MEMBRANE DAMAGE OF B CELL IN HAM/TSP

P075 Apoptosis in peripheral blood lymphocyte populations in HTLV-I infected people in vivo was examined. Freshly isolated lymphocytes from 10 non-infected healthy people, 8 asymptomatic HTLV-I carriers and 15 patients with HAM/TSP were stained with FITC-annexin-V to detect phosphatidylserine (PS) residue exposure at the outer plasma membrane leaflet as an early marker of apoptosis. There was no significant difference in annexin V positivity in CD4+ and CD8+ lymphocytes between 3 groups, but there was a greatly increased exposure of PS on CD19+ lymphocytes (B cells) in 12 out of 15 (80%) HAM/TSP patients, while only 2 out of 8 (25%) asymptomatic carriers and none of the non-infected healthy people showed this aberrant PS exposure on B cells. The intensity of annexin V staining of B cells in HAM/TSP was intermediate, as distinct from the high annexin V staining on advanced apoptotic cells. However, annexin V positivity was decreased when the cells were stained after 24 hours of culture, suggesting that the intermediate PS exposure on the B cell in HAM/TSP is not a consequence of the apoptotic process, but rather reflects a reversible membrane damage. B cells with PS exposure in vivo might provide a site for coagulation and inflammation, and so contribute to the pathogenesis of HAM/TSP and its complications.