Exogenous Phosphatidylcholine Research Articles

Loss of Opi3 causes a lipid imbalance that influences the virulence traits of Cryptococcus neoformans but not cryptococcosis.

The basidiomycete fungus Cryptococcus neoformans is a useful model for investigating mechanisms of fungal pathogenesis in mammalian hosts. This pathogen is the causative agent of cryptococcal meningitis in immunocompromised patients and is in the critical priority group of the World Health Organization fungal priority pathogens list. In this study, we employed a mutant lacking the OPI3 gene encoding a methylene-fatty-acyl-phospholipid synthase to characterize the role of phosphatidylcholine (PC) and lipid homeostasis in the virulence of C. neoformans. We first confirmed that OPI3 was required for growth in nutrient limiting conditions, a phenotype that could be rescued with exogenous choline and PC. Additionally, we established that loss of Opi3 and the lack of PC lead to an accumulation of neutral lipids in lipid droplets and alterations in major lipid classes. The growth defect of the opi3Δ mutant was also rescued by sorbitol and polyethylene glycol (PEG), a result consistent with protection of ER function from the stress caused by lipid imbalance. We then examined the impact of Opi3 on virulence and found that the dependence of PC synthesis on Opi3 caused reduced capsule size and this was accompanied by an increase in shed capsule polysaccharide and changes in cell wall composition. Further tests of virulence demonstrated that survival in alveolar macrophages and the ability to cause disease in mice were not impacted by loss of Opi3 despite the choline auxotrophy of the mutant in vitro. Overall, this work establishes the contribution of lipid balance to virulence factor elaboration by C. neoformans and suggests that host choline is sufficient to support proliferation during disease.

Reduced neuropathy target esterase in pre-eclampsia suppresses tube formation of HUVECs via dysregulation of phospholipid metabolism.

Recently, studies have shown that neuropathy target esterase (NTE) is essential to placental and normal blood vessel development. However, whether it is involved in abnormal placenta angiogenesis of pre-eclampsia remains unknown. Thus, our aim was to observe the expression of NTE in pre-eclamptic placentas and its effects and mechanism of NTE on the migration and the tube formation of human umbilical vein endothelial cells (HUVECs). Immunohistochemical staining showed that the NTE protein was intensely located in blood vessels of the normal pregnant placenta. However, western blot revealed that the expression level of NTE protein was significantly reduced in pre-eclamptic placenta. The results indicated that overexpression of NTE significantly promoted the migration and the tube formation of HUVECs compared with those of the control and scramble short hairpin RNA (shRNA) group. Conversely, NTE shRNA obviously inhibited the migration and the tube formation of HUVECs. Additionally, chromatography assay evidenced that NTE overexpression significantly reduced the level of phosphatidylcholine (PC) of HUVECs, but NTE shRNA obviously increased the level of PC of HUVECs. Furthermore, exogenous PC and lysophosphatidylcholine (LPC) significantly inhibited the tube formation of HUVECs in a dose-dependent manner. Collectively, our results suggest that reduced NTE in placenta may contribute to abnormal placenta angiogenesis of pre-eclampsia via the dysregulation of PC and LPC metabolism.

Exogenous phosphatidylcholine treatment alleviates drought stress and maintains the integrity of root cell membranes in peach

Phosphatidylcholine is a primary class of membrane lipids in most eukaryotes. In plants, the primary phosphatidylcholine biosynthetic pathway and its role in plant growth and development remain elusive due to lack of a mutant model with substantially decreased phosphatidylcholine content. In this study, we investigated whether the exogenous application of phosphatidylcholine can improve drought tolerance and reduce drought-induced damage to one-year-old peach (Prunus persica (L.) Batsch.). In this experiment, phosphatidylcholine was applied directly to the soil at concentrations of 200 mg/L, 500 mg/L, and 1000 mg/L to drought-stressed peach trees and at a concentration of 500 mg/L to control trees not subjected to drought stress. After these treatments, we assessed the net photosynthetic rate and chlorophyll content (i.e. SPAD values) of drought-stressed and unstressed trees. We found that trees sprayed with phosphatidylcholine resembled well-watered trees, but drought-stressed trees not supplemented with phosphatidylcholine showed a lower photosynthetic rate. In trees given sufficient water, the length and width of leaf stomata did not significantly differ regardless of whether plants were treated with phosphatidylcholine or not. However, untreated drought-stressed trees showed incompletely filled guard cells and low stomatal density relative to control trees. In addition, we also found that in drought-stressed trees exogenous phosphatidylcholine treatment resulted in significant increases in proline content as well as decreases in electrolyte leakage and the degree of damage to the cell membrane. Moreover, transmission electron microscopic observation of root cells of treated and untreated peach seedlings showed that phosphatidylcholine treatment protected the integrity of root cells in response to drought stress. Taken together, our results suggest that phosphatidylcholine treatment can improve the drought tolerance of young peach trees and can reduce the damage to cell membranes caused by drought stress.

Exogenous phosphatidylcholine supplementation retrieve aluminum-induced toxicity in male albino rats.

This study investigated the ameliorative potential of exogenous phosphatidylcholine (PC) against aluminum-induced toxicity in male albino rats. Four groups of rats were used for this study (N=8): group I served as the control, group II (PC treated) received L-α-phosphatidylcholine (egg yolk-derived) 100mg/kg bwt/day orally, group III (aluminum treated) received aluminum chloride 100mg/kg bwt/day orally, and group VI (aluminum+PC treated) received similar oral dose of aluminum and PC (100mg/kg bwt/day). Treatment was continued for 8weeks. Results revealed that aluminum chloride treatment leading to a significant elevation in serum aspartate aminotransferase, serum alanine aminotransferase, urea, creatinine, malondialdehyde, serum cytokines (tumor necrosis factor-α, interleukin-6), and brain content of acetylcholine, as well as a significant reduction in serum-reduced glutathione, serum testosterone, and brain content of acetylcholinesterase. Moreover, aluminum administration caused significant histopathological alteration in liver, kidney, brain, testes, and epididymis. Co-treatment with exogenous PC resulted in significant improvement in intensity of histopathologic lesions, serum parameters, testosterone level, proinflammatory cytokines, and oxidative/antioxidative status. However, it does not affect the brain content of acetylcholine and acetylcholinesterase. Conclusively, treatment with exogenous PC can retrieve the adverse effect of aluminum toxicities through its antioxidative and anti-inflammatory properties.

Exogenous phosphatidylcholine supplementation improves intestinal barrier defense against Clostridium difficile toxin.

The incidence and severity of Clostridium difficile colitis have increased dramatically in the last decade. Disease severity is related to C. difficile virulence factors, including toxins A and B, as well as the patient's immune status. The intestinal mucus is an important component of innate barrier function in the intestine. Phosphatidylcholine (PC) is a key constituent of the intestinal mucus barrier, and exogenous PC administration has had therapeutic efficacy in patients with ulcerative colitis. We studied the protective function of exogenous PC on C. difficile toxin effects on the intestinal barrier in vitro. Mucus-producing (HT29-MTX strain) and non-mucus-producing (HT29 strain) intestinal epithelial monolayers were cocultured with PC and C. difficile toxin A added to the apical media. Basal chamber culture supernatants were subsequently obtained, and tumor necrosis factor and interleukin 6 were quantitated by enzyme-linked immunosorbent assay. In other experiments, HT29 toxin A uptake, intestinal monolayer permeability, necrosis, and actin microfilament disruption were determined. There was a threefold to fourfold decrease in tumor necrosis factor and interleukin 6 levels and similar decreases in toxin A uptake and permeability changes in intestinal epithelial cells with mucus or PC versus control. Intestinal epithelial cell necrosis was reduced by more than 50% with either mucus or PC versus control. The integrity of HT29 cell cytoskeleton was demonstrated by both the mucus layer of the HT29-MTX strain and by exogenous PC administration by phalloidin staining of actin microfilaments. PC supplementation was effective in improving intestinal barrier defense against C. difficile toxin A challenge. PC administration may be a useful therapeutic adjunct in severe cases of C. difficile colitis or in patients who do not improve with conventional treatment.

Reinforcement of the intestinal mucus layer protects against Clostridium difficile intestinal injury in vitro.

Clostridium difficile infection is increasing in incidence and severity. Attributable factors include virulence factors, including C difficile toxins A and B, as well as host immunologic status. The mucus component of the intestinal barrier is impaired by malnutrition, shock insults, and alterations in the gut microbiome. Exogenous phosphatidylcholine (PC) administration results in reinforcement of the mucus layer and is of therapeutic benefit in chronic ulcerative colitis. We therefore studied the role of exogenous PC combined with secretory immunoglobulin A (IgA) in intestinal barrier function against C difficile infection invitro. Dimeric IgA was placed in the basal chambers of mucus-producing (HT29-methotrexate) and non-mucus-producing (HT29) strains of intestinal epithelial monolayers and allowed to undergo transcytosis and, in additional experiments, exogenous colostral IgA (30 ng/mL) was added to the apical media. After subsequent coculture with PC and C difficile toxin A in the apical chamber, tumor necrosis factor-α, interleukin-6, toxin A uptake, intestinal epithelial cell monolayer permeability, and necrosis were determined. A significant decrease of 4- to 5-fold in tumor necrosis factor-α and interleukin-6 levels and equally significant decreases in toxin A uptake and permeability changes in the intestinal cell monolayers with mucus or PC and transcytosed or colostral IgA vs control are shown. All groups analyzed also displayed a 2- to 3-fold reduction in necrosis. Mucus or "exogenous" mucus in the form of PC has a synergistic role with secretory IgA in barrier defense against C difficile toxin A. Exogenous PC administration can be a therapeutic adjunct in patients with severe or recalcitrant C difficile infection.

Advent of Novel Phosphatidylcholine-Associated Nonsteroidal Anti-Inflammatory Drugs with Improved Gastrointestinal Safety

The mucosa of the gastrointestinal (GI) tract exhibits hydrophobic, nonwettable properties that protect the underlying epithelium from gastric acid and other luminal toxins. These biophysical characteristics appear to be attributable to the presence of an extracellular lining of surfactant-like phospholipids on the luminal aspects of the mucus gel layer. Phosphatidylcholine (PC) represents the most abundant and surface-active form of gastric phospholipids. PC protected experimental rats from a number of ulcerogenic agents and/or conditions including nonsteroidal anti-inflammatory drugs (NSAIDs), which are chemically associated with PC. Moreover, preassociating a number of the NSAIDs with exogenous PC prevented a decrease in the hydrophobic characteristics of the mucus gel layer and protected rats against the injurious GI side effects of NSAIDs while enhancing and/or maintaining their therapeutic activity. Bile plays an important role in the ability of NSAIDs to induce small intestinal injury. NSAIDs are rapidly absorbed from the GI tract and, in many cases, undergo enterohepatic circulation. Thus, NSAIDs with extensive enterohepatic cycling are more toxic to the GI tract and are capable of attenuating the surface hydrophobic properties of the mucosa of the lower GI tract. Biliary PC plays an essential role in the detoxification of bile salt micelles. NSAIDs that are secreted into the bile injure the intestinal mucosa via their ability to chemically associate with PC, which forms toxic mixed micelles and limits the concentration of biliary PC available to interact with and detoxify bile salts. We have worked to develop a family of PC-associated NSAIDs that appear to have improved GI safety profiles with equivalent or better therapeutic efficacy in both rodent model systems and pilot clinical trials.

Use of phosphatidylcholine to improve the function of turkey semen stored at 4°C for 24 hours

The ability to store turkey semen for 24 h in vitro without a significant loss in fertility would benefit the commercial turkey industry. We investigated the use of exogenous phosphatidylcholine to improve the viability, mobility, hydrolyzing ability, and fertility of stored turkey sperm. For experiment 1, semen was diluted 1:1 with extender containing 0.0 (control), 0.5, 2.5, or 10.0 mg/mL of phosphatidylcholine labeled with a fluorochrome and maintained under aerobic conditions for 24 h at 4°C. Semen aliquots were removed at 30-min intervals during the first 4 h and at 1-h intervals from 8 to 24 h of storage for fluorometric evaluation by flow cytometry. Turkey sperm incorporated labeled phosphatidylcholine in a dose-dependent manner during the first 12 h of storage (P<0.05). At 24 h of storage, phosphatidylcholine uptake increased 7.8-fold for the 0.5-mg treatment, 9.2-fold for the 2.5-mg treatment, and 6.7-fold for the 10-mg labeled phosphatidylcholine treatment. For experiment 2, turkey semen was diluted and stored as for experiment 1 except the phosphatidylcholine was unlabeled. After 24 h, the viability, mobility, hydrolyzing ability, and fertility of turkey sperm was assessed. Supplemental phosphatidylcholine did not improve (P>0.05) the viability or mobility of stored sperm. At 2.5 mg/mL, phosphatidylcholine improved the hydrolyzing ability of stored sperm compared with control or other phosphatidylcholine treatments (P<0.05). The mean fertility rate of eggs from hens inseminated with control semen was 33.5%±4.5. Use of 0.5, 2.5, or 10.0 mg phosphatidylcholine/mL improved (P<0.05) the fertility rates during the first 11 wk of egg production; higher fertility rates occurred with 2.5 mg phosphatidylcholine/mL compared with other phosphatidylcholine treatments for 5 of those 11 wk (P<0.05). We conclude that supplemental phosphatidylcholine appears to counteract the damaging effects of lipid peroxidation or enzymatic degradation during in vitro storage by providing exogenous phospholipids for incorporation into the turkey sperm plasma membrane.

Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.

Depletion of phosphatidylcholine affects endoplasmic reticulum morphology and protein traffic at the Golgi complex

The mutant Chinese hamster ovary cell line MT58 contains a thermosensitive mutation in CTP:phosphocholine cytidylyltransferase, the regulatory enzyme in the CDP-choline pathway. As a result, MT58 cells have a 50% decrease in their phosphatidylcholine (PC) level within 24 h when cultured at the nonpermissive temperature (40 degrees C). This is due to a relative rapid breakdown of PC that is not compensated for by the inhibition of de novo PC synthesis. Despite this drastic decrease in cellular PC content, cells are viable and can proliferate by addition of lysophosphatidylcholine. By [(3)H]oleate labeling, we found that the FA moiety of the degraded PC is recovered in triacylglycerol. In accordance with this finding, an accumulation of lipid droplets is seen in MT58 cells. Analysis of PC-depleted MT58 cells by electron and fluorescence microscopy revealed a partial dilation of the rough endoplasmic reticulum, resulting in spherical structures on both sites of the nucleus, whereas the morphology of the plasma membrane, mitochondria, and Golgi complex was unaffected. In contrast to these morphological observations, protein transport from the ER remains intact. Surprisingly, protein transport at the level of the Golgi complex is impaired. Our data suggest that the transport processes at the Golgi complex are regulated by distal changes in lipid metabolism.

Anti-inflammatory effects of phosphatidylcholine in neutrophil leukocyte-dependent acute arthritis in rats

We investigated the effects of exogenous phosphatidylcholine (PC) and non-steroidal diclofenac supplementation on polymorphonuclear cell influx in carrageenan-induced arthritis in rats. The microcirculatory consequences were evaluated by a novel method developed for direct intravital microscopic observation of the synovial membrane. Arthritis was induced by injection of a mixture of 2% λ-carrageenan and 4% kaolin into the knee joints and the animals were treated orally with PC (150 mg/kg twice daily), sodium diclofenac (0.5 mg/kg twice daily) or saline vehicle. Intravital videomicroscopy was used to investigate the leukocyte-endothelial interactions directly in the synovial membrane at 6 h after the challenge. The inflammation–induced thermal and mechanical secondary hyperalgesic reactions were assessed at 24 h, and the knee volume changes at 48 h after the insult. The development of arthritis was accompanied by a significant increase in the number of adherent leukocytes in the synovial postcapillary venules, but this increase was reduced significantly (by ∼ 40%) by PC, and slightly (by 22%) by diclofenac treatment. The perivascular infiltration of the neutrophil leukocytes and the intercellular adhesion molecule-1 (ICAM-1) expressions were reduced only by PC treatment. The significant decrease (45%) in the thermal nociceptive latency, the 3-fold increase in the mechanical touch sensitivity and the knee cross-sectional area (which was increased by 35% by the arthritis induction) were significantly ameliorated by both treatments. The present study demonstrated the anti-inflammatory effects of PC in experimental arthritis. The therapeutic potential may be linked to the reduction of neutrophil leukocyte-mediated microcirculatory inflammatory reactions.

ORAL PHOSPHATIDYLCHOLINE PRESERVES THE GASTROINTESTINAL MUCOSAL BARRIER DURING LPS-INDUCED INFLAMMATION

The hydrophobic surface layer of the gastrointestinal (GI) tract, which has been attributed to the presence of phosphatidylcholine (PC) in the mucus gel, protects the mucosa of the GI tract and is disrupted by parenteral LPS treatment. We investigated the potential for repletion of this layer as a means to prevent LPS-induced GI injury. Rats were treated orally with PC 1 h before LPS (i.p.). Gastric and ileal tissues were assessed for changes in permeability 5 h later, and gastric fluid was analyzed for signs of GI-related LPS effects (bile acid reflux, increased volume, and pH) and gastric injury (bleeding). Serum TNF-alpha was assessed as a measure of a non-GI, LPS response. Radiolabeled PC was tracked through the GI tract to verify the extent of luminal exposure during the time of the study. Pretreatment with oral PC significantly blocked permeability increases in gastric and ileal tissue due to LPS. A portion of orally administered PC gained access to the entire GI tract in 1 h. Exogenous PC did not prevent the increase in serum TNF-alpha or gastric fluid volume or pH induced by LPS, nor did it prevent the duodenogastric reflux of bile acid. There was a tendency for PC to reduce gastric bleeding after LPS. Orally administered PC seems to act directly on the mucosa to prevent GI permeability disturbances due to LPS. Under the conditions studied, oral PC does not block systemic effects of LPS. However, enteral formulations containing PC may be useful adjuncts in the prevention of GI injury from endotoxemia.

Both Sphingomyelin Synthases SMS1 and SMS2 Are Required for Sphingomyelin Homeostasis and Growth in Human HeLa Cells

Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.

Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen–glucose deprivation in PC12 cells

Alterations in lipid metabolism play an integral role in neuronal death in cerebral ischemia. Here we used an in vitro model, oxygen–glucose deprivation (OGD) of rat pheochromocytoma (PC12) cells, and analyzed changes in phosphatidylcholine (PC) and sphingomyelin (SM) metabolism. OGD (4–8 h) of PC12 cells triggered a dramatic reduction in PC and SM levels, and a significant increase in ceramide. OGD also caused increases in phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD) activities and PLD2 protein expression, and reduction in cytidine triphosphate:phosphocholine cytidylyltransferase-α (CCTα, the rate-limiting enzyme in PC synthesis) protein expression and activity. Phospholipase A 2 activity and expression were unaltered during OGD. Increased neutral sphingomyelinase activity during OGD could account for SM loss and increased ceramide. Surprisingly, treatment with PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609) aggravated cell death in PC12 cells during OGD. D609 was cytotoxic only during OGD; cell death could be prevented by inclusion of sera, glucose or oxygen. During OGD, D609 caused further loss of PC and SM, depletion of 1,2-diacylglycerol (DAG), increase in ceramide and free fatty acids (FFA), cytochrome c release from mitochondria, increases in intracellular Ca 2+ ([Ca 2+] i), poly-ADP ribose polymerase (PARP) cleavage and phosphatidylserine externalization, indicative of apoptotic cell death. Exogenous PC during OGD in PC12 cells with D609 attenuated PC, SM loss, restored DAG, attenuated ceramide levels, decreased cytochrome c release, PARP cleavage, annexin V binding, attenuated the increase in [Ca 2+] i, FFA release, and significantly increased cell viability. Exogenous PC may have elicited these effects by restoring membrane PC levels. A tentative scheme depicting the mechanism of action of D609 (inhibiting PC-PLC, SM synthase, PC synthesis at the CDP-choline-1,2-diacylglycerol phosphocholine transferase (CPT) step and causing mitochondrial dysfunction) has been proposed based on our observations and literature.

Effect of phosphatidylcholine on postoperative adhesions after small bowel anastomosis in the rat.

The possibility of preventing postoperative adhesions using phosphatidylcholine (PC) was studied in rats. A small bowel anastomosis was created using continuous or interrupted 5/0 silk sutures. Adhesions were measured by recording the maximal length of adhesions around the anastomosis and the number of organs involved in the adhesions. PC was administered intraperitoneally either as a single dose or as three separate doses. Postoperative adhesions developed in all animals; the degree of adhesion was independent of the suture technique used. PC given once at a dose of 20 mg significantly decreased adhesion formation (P less than 0.01). The mechanism underlying this effect may be that the exogenous PC provides a lubricant layer replacing endogenous hydrolysed phospholipid. The result with repeated PC administration was not different from that after a single dose. Increasing the single dose of PC to 40 or 60 mg resulted in anastomotic dehiscence and subsequent peritonitis. It is concluded that PC in a single dose of 20 mg reduces adhesion formation after small bowel anastomosis in the rat.

Enhanced apoptosis through farnesol inhibition of phospholipase D signal transduction

Farnesol is a catabolite of the cholesterol biosynthetic pathway that preferentially causes apoptosis in tumorigenic cells. Phosphatidylcholine (PC), phosphatidic acid (PA), and diacylglycerol (DAG) were able to prevent induction of apoptosis by farnesol. Primary alcohol inhibition of PC catabolism by phospholipase D augmented farnesol-induced apoptosis. Exogenous PC was unable to prevent the increase in farnesol-induced apoptosis by primary alcohols, whereas DAG was protective. Farnesol-mediated apoptosis was prevented by transformation with a plasmid coding for the PA phosphatase LPP3, but not by an inactive LPP3 point mutant. Farnesol did not directly inhibit LPP3 PA phosphatase enzyme activity in an in vitro mixed micelle assay. We propose that farnesol inhibits the action of a DAG pool generated by phospholipase D signal transduction that normally activates an antiapoptotic/pro-proliferative target.

Enteropathogenic Escherichia coli Infection Triggers Host Phospholipid Metabolism Perturbations

Enteropathogenic Escherichia coli (EPEC) specifically recognizes phosphatidylethanolamine (PE) on the outer leaflet of host epithelial cells. EPEC also induces apoptosis in epithelial cells, which results in increased levels of outer leaflet PE and increased bacterial binding. Consequently, it is of interest to investigate whether EPEC infection perturbs host cell phospholipid metabolism and whether the changes play a role in the apoptotic signaling. Our findings indicate that EPEC infection results in a significant increase in the epithelial cell PE level and a corresponding decrease in the phosphatidylcholine (PC) level. PE synthesis via both the de novo pathway and the serine decarboxylation pathway was enhanced, and de novo synthesis of phosphatidylcholine via CDP-choline was reduced. The changes were transitory, and the maximum change was noted after 4 to 5 h of infection. Addition of exogenous PC or CDP-choline to epithelial cells prior to infection abrogated EPEC-induced apoptosis, suggesting that EPEC infection inhibits the CTP-phosphocholine cytidylyltransferase step in PC synthesis, which is reportedly inhibited during nonmicrobially induced apoptosis. On the other hand, incorporation of exogenous PE by the host cells enhanced EPEC-induced apoptosis and necrosis without increasing bacterial adhesion. This is the first report that pathogen-induced apoptosis is associated with significant changes in PE and PC metabolism, and the results suggest that EPEC adhesion to a host membrane phospholipid plays a role in disruption of host phospholipid metabolism.

Phospholipase A 2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver

The present study is undertaken to investigate whether the phospholipase A 2 (PLA 2) influences mRNA nucleocytoplasmic transport evaluated by nucleoside triphosphatase (NTPase) activity and mRNA export in isolated hepatic nuclear envelope. Isolated hepatic nuclei from rat liver were exposed to PLA 2 (10 −5 ∼ 10 −2/ml) with or without incorporation of nuclei with phosphatidylcholine (PC) liposome. Messenger RNA exports and NTPase activities of nuclear membrane were assayed using ATP and GTP as substrates. We found that the RNA efflux, evaluated by [ 3H] uridine, was potently decreased in a concentration-dependent manner, by incubation of hepatic nuclei with PLA 2, regardless using ATP or GTP as substrates. The PC content in nuclear membrane was also decreased by PLA 2-treatment. The PC was incorporated into the nuclear membrane by addition of phospholipid liposomes into the incubation mixture. PC incorporation into the nuclear membrane did not alter mRNA export. However this resulted in a significant increase in mRNA export rate in PLA 2-treated group. Messenger RNA export rate in PLA 2 (10 −3 unit/mL)- treated nuclear membrane was positively correlated with level of PC incorporation, both using ATP and GTP as substrates. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme, showed parallel variations with mRNA transport. It is concluded that nuclear PLA 2 plays a regulatory role in RNA transport, which can be antagonized by exogenous PC. These might be pathophysiologically significance, although the mechanisms by which this effect takes place remain to be clarified.

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora.

Exogenous fluorescent phosphatidic acid (PA) and phosphatidylcholine (PC) were transported into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora [Kamisaka et al. (1999) Biochim. Biophys. Acta 1438, 185-198]. We further investigated the processes of fluorescent PA and PC transport into lipid bodies in this fungus by changing culture conditions. Lowering incubation temperature decreased lipid body labeling by 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-PA (C5-DMB-PA), but fluorescence did not accumulate in organelles other than lipid bodies. C5-DMB-PC transport into lipid bodies was blocked at temperatures below 15 degrees C and fluorescence accumulated in intracellular membranes, presumably endoplasmic reticulum membranes. The low-temperature block of C5-DMB-PC transport enabled us to do pulse-chase experiments in which fungal cells were pulse-labeled at 15 degrees C with C5-DMB-PC and chased at 30 degrees C. The results clearly depicted transport of C5-DMB-PC and its derivatives from intracellular membranes to lipid bodies. Transport was temperature-dependent and ATP-dependent, although microtubules and actin filaments were not substantially involved. Experiments using 14C-labeled fatty acids and glycerol instead of C5-DMB-PC under the same conditions suggested that transport depicted by fluorescence agreed with metabolism and transport of PC containing native fatty acids. Furthermore, the transport mechanism preferred PC containing unsaturated fatty acids such as linoleic acid. This study dissect lipid transport of PA and PC into lipid bodies and reveal regulatory steps for lipid body formation in this fungus.

Inhibition of phosphatidylcholine synthesis precedes apoptosis induced by C2-ceramide

Cerebellar granule neurons in primary culture underwent apoptosis when exposed to C2-ceramide. Addition of exogenous phosphatidylcholine (PtdCho) resulted in a dose-dependent full prevention of neuronal death. Exogenous PtdCho also prevented apoptosis induced by farnesol, N-oleoylethanolamine, and sphingomyelinase, but did not prevent apoptosis induced after lowering the potassium concentration in the medium to non-depolarizing levels. Moreover, C2-ceramide inhibited labeling of [32P]PtdCho in cells incubated with [32P]orthophosphate, with the same potency to that causing apoptosis. Although cell viability did not decrease during the first few hours, inhibition of PtdCho synthesis was already patent after a 1 h exposure to C2-ceramide. Taken together, these results strongly suggest that inhibition of PtdCho synthesis constitutes one of the primary events by which C2-ceramide triggers apoptosis in cerebellar granule neurons.