Nucleated cell killing by complement: effects of C5b-9 channel size and extracellular Ca2+ on the lytic process

For C5b-9 channels to mediate cytolysis of a nucleated cell, a sufficient number of channels must be formed in the plasma membrane to override the compensatory mechanisms that nucleated cells might employ to survive. It is well known that nucleated cells are relatively resistant to lysis by complement in comparison to erythrocytes, and it is now evident that this resistance is due, in part, to the ability of nucleated cells to rapidly eliminate C5b-9 from the cell surface. The ability of nucleated cells to eliminate complement complexes is related to physiochemical properties of the complex, such as channel diameter, which in turn affect Ca2+ fluxes that stimulate metabolic processes involved in the elimination process. Paradoxically, these same channel properties that stimulate the defense response may also be responsible for the lethal effects of complement. To further study the role of channel size on cytolysis of nucleated cells by C5b-9, we examined the lytic efficiency of larger C5b-9 channels containing several C9 molecules in comparison with smaller C5b-9 channels containing fewer C9. We have obtained data to indicate that although the larger channels were more cytolytically potent, the channel size had little influence on the rate of cell death. In contrast, the rate of lysis of erythrocytes was substantially slower when smaller C5b-9 channels were present. In evaluating the effect of the extracellular Ca2+ concentration, [Ca2+]o, on nucleated cell lysis in the presence of a lytic number of C5b-9 complexes, it was observed that when the [Ca2+]o was increased the rate of cell death also increased. These findings suggest that lysis of nucleated cells by C5b-9, unlike erythrocytes, may not be entirely due to colloid osmotic deregulation.     

Intracellular Ca and cell injury: A paradoxical role of Ca in complement membrane attack

Disturbances in intracellular Ca2+ are known to be important in cell injury caused by a wide range of toxic factors. The complement system is a major effector of immune damage in vivo, and is known to be involved in the pathogenesis of many immune diseases. We present here evidence that the potentially lethal membrane attack complex of complement causes a rapid increase in intracellular free Ca2+ concentration before any other detectable biochemical changes in the cell. In nucleated cells the increased intracellular free Ca2+ concentration initially stimulates recovery processes, allowing the cell to escape mild complement attack and also activates the production of inflammatory mediators, which may amplify an ongoing inflammatory response. More severe complement membrane attack causes a more rapid rise in intracellular free Ca2+ concentration allowing a threshold to be breached above which recovery processes are overwhelmed, and cell death occurs. The importance of non-lytic effects and recovery processes mediated by Ca2+, and the molecular basis of these effects are discussed, and the hypothesis proposed that the cell-injuring effects of other "pore-forming" toxins are also caused by increases in intracellular free Ca2+.     

On the pathogenesis of galactosamine hepatitis. Indications of extrahepatocellular mechanisms responsible for liver cell death.

In order to evaluate the pathogenesis of galactosamine hepatitis, the action of galactosamine on mast cells, and alteration in the complement system suring the course of this experimental injury were studied. It has been previously demonstrated that rat livers after colectomy are refractory to galactosamine-induced liver cell necrosis and inflammation. For this reason colectomized animals were used to see whether the biochemical alterations produced by this aminosugar and thought to be responsible for cell death developed. Results showed: 1. galactosamine potently degranulates mast cells in vivo and in vitro, 2. the complement system is a) activated during the course of galactosamine hepatitis, probably by circulating endotoxins, and b) is essential for liver cell death in galactosamine hepatitis, and 3. colectomy does not prevent biochemical changes known to occur during galactosamine metabolism. It is concluded that death of galactosamine-injured liver cells is triggered by extrahepatocellular mechanisms, which lead ultimately to an activated complement system by endotoxins. It is postulated that related mechanism may also occur in viral hepatitis and in fulminant hepatic failure in man.     

Apoptosis and eryptosis: Striking differences on biomembrane level

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis. In contrast to nucleated cells, in which a significant drop of the lipid order in the plasma membrane is observed, the erythrocyte membrane retains the relatively high level of the lipid order. Observation in nucleated cells of significant differences between inner and plasma membranes and detection of apoptotic bodies with different organization suggest that the decrease in the lipid order of their plasma membrane could be at least partially explained by the phospholipid and/or cholesterol exchange between membranes. Such features are absent in erythrocytes.     

Role of acetoin on the regulation of intermediate metabolism of Ehrlich ascites tumor mitochondria: its contribution to membrane cholesterol enrichment modifying passive proton permeability

Acetoin, an unusual metabolite of highly glycolytic mammalian tumor cells, is synthesized from decarboxylated pyruvate and active acetaldehyde in mitochondria. It plays important roles in the regulation and detoxification of pyruvate metabolism through pyruvate dehydrogenase. We show in this report the inhibitory effect of acetoin on succinate oxidation by Ehrlich tumor cell mitochondria, and thus its regulatory role on intermediate metabolism. Acetoin utilization by Ehrlich mitochondria may lead to small quantities of citrate formation which increase the already increased cholesterol synthesis of cancer cells. Membranes, in particular the inner mitochondrial membrane, flooded with cholesterol, show a proton passive permeability twice as low as that of control mitochondrial membranes, a feature that may be related to drastic changes in membrane potential-dependent metabolism of cancer cells.     

Intrinsic resistance of hepatocytes to complement-mediated injury

When activated on or in the vicinity of cells, complement usually causes loss of function and sometimes cell death. Yet the liver, which produces large amounts of complement proteins, clears activators of complement and activated complexes from portal blood without obvious injury or impaired function. We asked whether and to what extent hepatocytes resist injury and loss of function mediated by exposure to complement. Using cells isolated from porcine livers as a model system, we found that, in contrast to endothelial cells, hepatocytes profoundly resist complement-mediated lysis and exhibit normal synthetic and conjugative functions when complement is activated on their surface. The resistance of hepatocytes to complement-mediated injury was not a function of cell surface control of the complement cascade but rather an intrinsic resistance of the cells dependent on the PI3K/Akt pathway. The resistance of hepatocytes to complement might be exploited in developing approaches to the treatment of hepatic failure or more broadly to the treatment of complement-mediated disease.     

Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion

Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.     

Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase is sensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODs used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [14C]acetate incorporation into [14C]cholesterol and by an increased [125I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.     

Structural and functional changes in erythrocyte membranes in experimental atherosclerosis

Structural and functional characteristics of erythrocyte membranes were studied in rabbits with experimental atherosclerosis. In animals with single lipid spots in the aorta, a significant rise of the plasma cholesterol level was associated with the increased cholesterol/phospholipid (CS/PL) ratio and diminished activity of erythrocyte membrane Na+, K+-ATPase. EPMR spin probe data point to changes in structural membrane characteristics--an increase in order parameter for fatty acid chains of lipids and expansion of the temperature interval of the transition phase in the membranes. In rabbits with total aorta injury, a further increase both in the plasma cholesterol concentration and in the CS/PL ratio as well as in structural changes in erythrocyte membranes does not lead to another decrease in the enzymatic activity. In aorta homogenates of the experimental animals, the activity of Na+, K+-ATPase correlated with that in the erythrocyte membrane. This suggests the existence of similar chemical and structural changes in aorta cell membranes. The data may provide an indirect evidence in favour of the hypothesis of the involvement of smooth muscle cells and membrane enzymatic activity alterations in atherosclerosis.     

Two different pathways for necrotic cell death induced by free radicals

Plasma membrane modifications have been widely recognized as crucial factors in cell injury and death. One of these modifications, surface blebbing, has been considered as an injury-marker associated with a series of biochemical and physiological modifications. Our study focused on the different effects of free radical-induced cell damage by quinone menadione (2-methyl-1,4-naphthoquinone) and by hyperthermic shock (45°C) on the erythroleukemic cell line K.562. Different techniques including immunofluorescence, freeze-fracturing, and electron paramagnetic resonance spectroscopy were employed. Menadione induced the formation of surface blebs, accompanied by a rearrangement of the microfilament system and changes in the distribution of plasma membrane proteins. In contrast, heat-shocked cells showed neither blebbing nor important cytoskeletal changes. Finally, the electron paramagnetic resonance results showed an increase in membrane order not specifically related to the type of free radical-induced stress. These cell death features appear to suggest the existence of two different types ofpathways for necrotic cell death: both treatments induce cell injury and eventual death by modifiting plasma membrane integrity and function. However, one involves cytoskeleton-dependent surface blebbing, whereas the other does not.Abbreviations EPR electron paramagnetic resonance - HS heat shock - IMPs intramembrane particles - MEN menadione     

Pulsatilla chinensis saponins cause liver injury through interfering ceramide/sphingomyelin balance that promotes lipid metabolism dysregulation and apoptosis

Background: P. chinensis saponins (PRS) are pentacyclic triterpenoid bioactive constituents from Pulsatilla chinensis (Bunge) Regel. In our previous study, PRS caused chronic liver injury (CLI) with the significant changes of lipid metabolites including sphingomyelin (SM) in serum after long-term administration. The SM in the hepatocytes membrane plays an indispensable role in maintaining cell membrane stability and regulating the extracellular and intracellular signal transduction. However, it is still unknown the pathway related to SM and the mechanism of CLI on hepatocyte.Purpose: The purpose of this study was to explore the hepatotoxicity mechanism of PRS in vivo and in vitro, to reveal the action of mechanism of SM and the pathway related to liver injury.Methods: SD rats were orally administered with PRS for 240 days and liver injury was evaluated by histological examinations. Metabolomics analysis was used to explore the liver metabolic pathway affected by PRS, and the expressions of related proteins were evaluated by western blots. To discover and elucidate the underlying mechanisms of metabolites changes induced by PRS at the cellular level, cellular morphology, MTT assays, western blots and cell membrane potential measurements were carried out using LO2 cells. Furthermore, the roles of SM and cholesterol (Chol) in hepatocyte injury were investigated individually in overload Chol and SM groups. Sphingolipid metabolic pathway related with ceramide/sphingomyelin (Cer/SM) balance was explored using cellular lipidomics and RT-PCR.Results: PRS gradually damaged the rat's liver in a time-dependent manner. The analysis of liver metabolism profiles showed that lipids metabolites were changed, including sphingolipid, bile acid, linoleic acid and fatty acid. We found that PRS induced apoptosis by interfering with bile acid-mediated sphingolipid metabolic pathway and Cer/SM balance in CLI. In in vitro experiments, PRS led to the increase of LDH leakage, depolarized cell membrane potential and caused cell membrane toxicity. Furthermore, PRS inducedG0/G1 phase cell cycle arrest in LO2 cells, simultaneously activated cellular extrinsic and intrinsic apoptosis pathways. PRS acted on SM and interfered with Cer/SM balance, which promote lipid metabolism dysregulation and apoptosis.Conclusion: PRS acted on SM to interfere Cer/SM balance on LO2 cell. Both in vivo and in vitro, PRS induced Cer/SM imbalance which promoted lipid metabolism disorder and apoptosis. Apoptosis and lipids changes gradually damaged the rats liver, and ultimately developed into CLI.     

Caveolin-1 and dynamin-2 are essential for removal of the complement C5b-9 complex via endocytosis

The complement system, an important element of both innate and adaptive immunity, is executing complement-dependent cytotoxicity (CDC) with its C5b-9 protein complex that is assembled on cell surfaces and transmits to the cell death signals. In turn, cells, and in particular cancer cells, protect themselves from CDC in various ways. Thus, cells actively remove the C5b-9 complexes from their plasma membrane by endocytosis. Inhibition of clathrin by transfection with shRNA or of EPS-15 with a dominant negative plasmid had no effect on C5b-9 endocytosis and on cell death. In contrast, inhibition of caveolin-1 (Cav-1) by transfection with an shRNA or a dominant negative plasmid sensitized cells to CDC and inhibited C5b-9 endocytosis. Similarly, both inhibition of dynamin-2 by transfection with a dominant negative plasmid or by treatment with Dynasore reduced C5b-9 endocytosis and enhanced CDC. C5b-9 endocytosis was also disrupted by pretreatment of the cells with methyl-β-cyclodextrin or Filipin III, hence implicating membrane cholesterol in the process. Analyses by confocal microscopy demonstrated co-localization of Cav-1-EGFP with C5b-9 at the plasma membrane, in early endosomes, at the endocytic recycling compartment and in secreted vesicles. Further investigation of the process of C5b-9 removal by exo-vesiculation demonstrated that inhibition of Cav-1 and cholesterol depletion abrogated C5b-9 exo-vesiculation, whereas, over-expression of Cav-1 increased C5b-9 exo-vesiculation. Our results show that Cav-1 and dynamin-2 (but not clathrin) support cell resistance to CDC, probably by facilitating purging of the C5b-9 complexes by endocytosis and exo-vesiculation.     

Targeting mitochondria-associated membranes as a potential therapy against endothelial injury induced by hypoxia

Mitochondrial dysfunction plays a principal role in hypoxia-induced endothelial injury, which is involved in hypoxic pulmonary hypertension and ischemic cardiovascular diseases. Recent studies have identified mitochondria-associated membranes (MAMs) that modulate mitochondrial function under a variety of pathophysiological conditions such as high-fat diet-mediated insulin resistance, hypoxia reoxygenation-induced myocardial death, and hypoxia-evoked vascular smooth muscle cell proliferation. However, the role of MAMs in hypoxia-induced endothelial injury remains unclear. To explore this further, human umbilical vein endothelial cells and human pulmonary artery endothelial cells were exposed to hypoxia (1% O₂) for 24 hours. An increase in MAM formation was uncovered by immunoblotting and immunofluorescence. Then, we performed small interfering RNA transfection targeted to MAM constitutive proteins and explored the biological effects. Knockdown of MAM constitutive proteins attenuated hypoxia-induced elevation of mitochondrial Ca²⁺ and repressed mitochondrial impairment, leading to an increase in mitochondrial membrane potential and ATP production and a decline in reactive oxygen species. Then, we found that MAM disruption mitigated cell apoptosis and promoted cell survival. Next, other protective effects, such as those pertaining to the repression of inflammatory response and the promotion of NO synthesis, were investigated. With the disruption of MAMs under hypoxia, inflammatory molecule expression was repressed, and the eNOS-NO pathway was enhanced. This study demonstrates that the disruption of MAMs might be of therapeutic value for treating endothelial injury under hypoxia, suggesting a novel strategy for preventing hypoxic pulmonary hypertension and ischemic injuries.     

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.     

Squalene synthase, a determinant of Raft-associated cholesterol and modulator of cancer cell proliferation

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.     

LPCAT1 reprogramming cholesterol metabolism promotes the progression of esophageal squamous cell carcinoma

Tumor cells require high levels of cholesterol for membrane biogenesis for rapid proliferation during development. Beyond the acquired cholesterol from low-density lipoprotein (LDL) taken up from circulation, tumor cells can also biosynthesize cholesterol. The molecular mechanism underlying cholesterol anabolism in esophageal squamous cell carcinoma (ESCC) and its effect on patient prognosis are unclear. Dysregulation of lipid metabolism is common in cancer. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been implicated in various cancer types; however, its role in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we identified that LPCAT1 is highly expressed in ESCC and that LPCAT1 reprograms cholesterol metabolism in ESCC. LPCAT1 expression was negatively correlated with patient prognosis. Cholesterol synthesis in ESCC cells was significantly inhibited following LPCAT1 knockdown; cell proliferation, invasion, and migration were significantly reduced, along with the growth of xenograft subcutaneous tumors. LPCAT1 could regulate the expression of the cholesterol synthesis enzyme, SQLE, by promoting the activation of PI3K, thereby regulating the entry of SP1/SREBPF2 into the nucleus. LPCAT1 also activates EGFR leading to the downregulation of INSIG-1 expression, facilitating the entry of SREBP-1 into the nucleus to promote cholesterol synthesis. Taken together, LPCAT1 reprograms tumor cell cholesterol metabolism in ESCC and can be used as a potential treatment target against ESCC.Subject terms: Cancer metabolism, Cancer prevention     

Effect of drugs which inhibit cholesterol synthesis on syncytia formation in vero cells infected with measles virus

We found that nontoxic doses of two inhibitors of cholesterol synthesis, namely W-7 and cerulenin, delayed syncytia formation in vero cells infected with measles virus. To correlate syncytia formation and lipidic membrane changes induced by these drugs, we labelled cell lipids with [14C]acetate. Measles virus infection increased the incorporation of radiolabel into fatty acids, triacylglycerol, cholesterol ester, and decreased its incorporation into cholesterol and 1,2-diacylglycerol. The ratios phosphatidylcholine/sphingomyelin and free cholesterol/lanosterol-dihydrolanosterol also decreased during the infection. W-7 and cerulenin greatly altered lipid metabolism. Both decreased the phosphatidylcholine to sphingomyelin and the cholesterol to lanosterol-dihydrolanosterol ratios. Z-D-Phe-L-Phe-L-Gly, a tripeptide which corresponds to the N-terminal sequence of the viral fusion protein (responsible for syncytia formation) and which inhibits virus-induced cell fusion without affecting virus synthesis also perturbed cholesterol metabolism. The tripeptide reversed the phosphatidylcholine to sphingomyelin ratio in infected cells. At non-toxic doses, W-7 inhibited the synthesis of infectious virus. Cerulenin which inhibited strongly the lipid synthesis did not. Finally, the well characterized inhibitors of cholesterol synthesis, mevinolin, ketoconazole and miconazole were shown to inhibit the syncytia formation. We conclude that the inhibition of syncytia by W-7 and cerulenin is associated with their capacity to alter the cholesterol metabolism, whereas the antiviral effect of W-7 does not seem related to this capacity.     

Effects of High Pressure on Survival and Metabolic Activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10⁶ cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.