Phospholipid Asymmetry Research Articles

Role of C. elegans TAT-1 Protein in Maintaining Plasma Membrane Phosphatidylserine Asymmetry

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.

Mechanism of erythrocyte death in human population exposed to arsenic through drinking water

Arsenic contamination in drinking water is one of the biggest natural calamities, which has become an imperative threat to human health throughout the world. Abbreviation of erythrocyte lifespan leading to the development of anemia is a common sequel in arsenic exposed population. This study was undertaken to explore the mechanism of cell death in human erythrocytes during chronic arsenic exposure. Results revealed transformation of smooth discoid red cells into evaginated echinocytic form in the exposed individuals. Further distortion converted reversible echinocytes to irreversible spheroechinocytes. Arsenic toxicity increased membrane microviscosity along with an elevation of cholesterol/phospholipid ratio, which hampered the flexibility of red cell membrane and made them less deformable. Significant increase in the binding of merocyanine 540 with erythrocyte membrane due to arsenic exposure indicated disruption of lipid packing in the outer leaflet of the cell membrane resulting from altered transbilayer phospholipid asymmetry. Arsenic induced eryptosis was characterized by cell shrinkage and exposure of phosphatidylserine at the cell surface. Furthermore, metabolic starvation with depletion of cellular ATP triggered apoptotic removal of erythrocytes from circulation. Significant decrease in reduced glutathione content indicating defective antioxidant capacity was coupled with enhancement of malondialdehyde and protein carbonyl levels, which pointed to oxidative damage to erythrocyte membrane. Arsenic toxicity intervened into red cell membrane integrity eventually leading to membrane destabilization and hemoglobin release. The study depicted the involvement of both erythrophagocytosis and hemolysis in the destruction of human erythrocytes during chronic arsenic exposure.

Chemoprotective Action of Resveratrol and Genistein From Apoptosis Induced in Human Peripheral Blood Lymphocytes

Extensive research is being carried out to analyse the importance of plant products such as resveratrol and genistein, which are known to exert a variety of pharmacological effects. This study aims at evaluating the protective role of these compounds against the apoptosis induced in normal cells by cytotoxic anticancer agents such as cisplatin and mytomycin C during therapy. Despite the broad antineoplastic action of cisplatin and mitomycin C, their genotoxicity in normal cell might lead to the induction of secondary malignancies. Therefore, the problem of identifying plant compounds, which might exert protective action in normal cells, gains lot of significance. We have analyzed the chemoprotective effect of plant compounds on peripheral blood human lymphocytes when exposed to cisplatin and mitomycin C by pre-treating and post-treating them with resveratrol and genistein at 100 μM concentration Biochemical alterations occurring in many cells during apoptosis include loss of plasma membrane phospholipid asymmetry, DNA fragmentation, and activation of caspase-3, et cetera, and have been assessed. Fluorescence microscopy, flow cytometric techniques have clearly demonstrated that resveratrol and genistein are efficient in protecting lymphocytes undergoing DNA damage when exposed to cisplatin and mitomycin C and exerted their activity by reducing the caspase 3 expression. An interesting observation is that, these compounds offered their protective effect by reducing their apoptotic potential on membrane and nucleic acids against cytotoxic agents, cisplatin, and mitomycin C. These results suggest that resveratrol and genistein might be useful for risk assessments in advance of clinical trials and could be considered as a strong candidate in pharmacogenomics or nutriprotective arena.

Protein kinases Fpk1p and Fpk2p are novel regulators of phospholipid asymmetry.

Type 4 P-type ATPases (flippases) are implicated in the generation of phospholipid asymmetry in membranes by the inward translocation of phospholipids. In budding yeast, the DRS2/DNF family members Lem3p-Dnf1p/Dnf2p and Cdc50p-Drs2p are putative flippases that are localized, respectively, to the plasma membrane and endosomal/trans-Golgi network (TGN) compartments. Herein, we identified a protein kinase gene, FPK1, as a mutation that exhibited synthetic lethality with the cdc50Delta mutation. The kinase domain of Fpk1p exhibits high homology to plant phototropins and the fungus Neurospora crassa NRC-2, both of which have membrane-associated functions. Simultaneous disruption of FPK1 and its homolog FPK2 phenocopied the lem3Delta/dnf1Delta dnf2Delta mutants, exhibiting the impaired NBD-labeled phospholipid uptake, defects in the early endosome-to-TGN pathway in the absence of CDC50, and hyperpolarized bud growth after exposure of phosphatidylethanolamine at the bud tip. The fpk1Delta fpk2Delta mutation did not affect the subcellular localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Further, the purified glutathione S-transferase (GST)-fused kinase domain of Fpk1p phosphorylated immunoprecipitated Dnf1p and Dnf2p to a greater extent than Drs2p. We propose that Fpk1p/Fpk2p are upstream activating protein kinases for Lem3p-Dnf1p/Dnf2p.

Quality control of blood irradiation with a teletherapy unit: damage to stored red blood cells after cobalt‐60 gamma irradiation

Previous publications have documented the damage caused to red blood cells (RBCs) irradiated with X-rays produced by a linear accelerator and with gamma rays derived from a 137Cs source. The biologic effects on RBCs of gamma rays from a 60Co source, however, have not been characterized. This study investigated the effect of 3000 and 4000 cGy on the in vitro properties of RBCs preserved with preservative solution and irradiated with a cobalt teletherapy unit. A thermal device equipped with a data acquisition system was used to maintain and monitor the blood temperature during irradiation. The device was rotated at 2 r.p.m. in the irradiation beam by means of an automated system. The spatial distribution of the absorbed dose over the irradiated volume was obtained with phantom and thermoluminescent dosimeters (TLDs). Levels of Hb, K+, and Cl(-) were assessed by spectrophotometric techniques over a period of 45 days. The change in the topology of the RBC membrane was investigated by flow cytometry. Irradiation caused significant changes in the extracellular levels of K+ and Hb and in the organizational structure of the phospholipid bilayer of the RBC membrane. Blood temperature ranged from 2 to 4 degrees C during irradiation. Rotation at 2 r.p.m. distributed the dose homogeneously (92%-104%) and did not damage the RBCs. The method used to store the blood bags during irradiation guaranteed that all damage caused to the cells was exclusively due to the action of radiation at the doses applied. It was demonstrated that prolonged storage of 60Co-irradiated RBCs results in loss of membrane phospholipids asymmetry, exposing phosphatidylserine (PS) on the cells' surface with a time and dose dependence, which can reduce the in vivo recovery of these cells. A time- and dose-dependence effect on the extracellular K+ and plasma-free Hb levels was also observed. The magnitude of all these effects, however, seems not to be clinically important and can support the storage of irradiated RBC units for at last 28 days.

Oxidative Stress Induces Changes and Shedding of Membrane Phospholipids from Thalassemic RBCs - A NMR Study.

Changes in the membrane phospholipid (PL) asymmetry is one of the hallmarks of apoptosis in nucleated cells. Although mature anucleated RBCs, do not undergo the classical pattern of apoptosis, upon trauma or aging they present changes in the membrane asymmetry. These changes include a flip-flop of phosphatidylserine (PS) from the inner leaflet of the membrane to its outer leaflet. This externalization of PS stimulates RBC phagocytosis by macrophages of the reticulo-endothelial system and their removal from the circulation. Oxidative stress is among the causes of PS externalization on RBCs. In beta-thalassemia and sickle cell disease, although the primary defects are mutations in the globin genes, oxidative stress is thought to mediate part of the damage to the RBCs, and particularly to its membrane, including PS externalization. In the present study, we used Nuclear Magnetic Resonance (NMR) spectroscopy to analyze normal and beta-thalassemic RBCs in order to study the relationship between their oxidative status and the content and shedding of their PL. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio in thalassemic RBCs, confirming their state of oxidative stress. Using 31P-NMR, we measured the content of various PLs, and found 30±3% more phosphatidylcholine (PC), and unexpectedly, less PS in thalassemic RBCs than in normal RBCs. The PS was increased in thalassemic RBC, but not in normal RBC, by treatment with anti-oxidants (vitamin C and N-acetyl cysteine) and decreased by oxidants (t-butylhydroxyperoxide and H2O2) in normal and thalassemic RBCs. PC showed the opposite behavior, indicating a correlation between PS and PC contents and the oxidative status. Since RBCs with exposed PS have been reported to be more frequent in thalassemic blood than in normal blood, we hypothesized that the decrease in PS is a result of shedding from the external membrane, either as free PS moieties or as part of membrane vesicles. NMR analysis of blood plasma obtained from normal and thalassemic donors indicated a 2.6-fold and 1.8-fold increase in PS and PC, respectively in the latter plasma. In vitro incubation of RBC produced much higher PS in supernatants derived from thalassemic RBCs compared with those of normal RBCs. Anti-oxidants reduced the PS shedding from thalassemic RBCs into their supernatants while oxidants increased the PS shedding by normal RBCs. RBCs are known to shed membranous particles (termed vesicles or microparticles) in vitro and in vivo during their physiologic and pathological senescence. We studied this point by purifying microparticles from plasma and RBC supernatants of normal and thalassemic donors, and measuring the PLs content in their lipophilic extracts by 31P-NMR. We found that the PS content and its proportion out of the total PLs were higher in microparticles purified from thalassemic plasma (0.25±0.04 mM, 19% of the plasma total PS) or RBC supernatants than in normal plasma microparticles (0.045±0.06 mM, 9.5% of the plasma total PS) or supernatants. The results also show that although microparticles are enriched in PS compared to their intact RBCs, the bulk of the shed PS is not associated with microparticles. These results suggest that oxidative stress in RBCs causes them to shed their PS and that the increase in PC levels maybe be a compensating mechanism. The pathological consequences of these phenomena on the survival of RBCs in thalassemia warrants further study.

External PS in Sickle RBC: Relationships among Aminophospholipid Translocase (APLT), Phospholipid Scramblase (PLSCR), Cell Maturity, and Cell Density.

External phosphatidylserine (PS) is present on some sickle RBC and may contribute to thrombogenesis, endothelial adhesion, and shortened RBC lifespan. Phospholipid scramblase (PLSCR) disrupts phospholipid (PL) asymmetry by causing nonspecific PL equilibration across the membrane. Aminophospholipid translocase (APLT) maintains PL asymmetry by returning externalized PS to the inner membrane leaflet. It has been proposed that both APLT inhibition and PLSCR activation are required for PS externalization. Sickle RBC with low level external PS (Type I PS+) are present in cells of all densities and include some reticulocytes. Sickle RBC with high external PS (Type II PS+) are primarily found in the dense fraction. Type II cells are thought to be more important because:1.the high level of external PS should have greater consequence;2.high level external PS occurs primarily in pathologically dehydrated sickle RBC; and3.low level external PS appears to be physiological in immature RBC.We have previously shown that dense, dehydrated sickle RBC, including the small number of dense transferrin receptor positive (TfR+) reticulocytes, have markedly inhibited APLT. In the current studies, we examined the relationships among external PS, APLT, PLSCR, and density in mature RBC and TfR+ reticulocytes using 3-color flow cytometry. APLT and PLSCR activities were assayed using fluorescent PL analogues (NBD-PS and NBD-PC, respectively), and expressed as the fraction of probe internalized. External PS was measured with Annexin V-PE and TfR+ reticulocytes were identified with anti-TfR-PE/Cy5. PS+ cells had lower APLT activity compared to PS- cells that did not reach significance for n=3 (NBD-PS internalization fraction for PS-: 0.586±0.053; Type I PS+: 0.517±0.158, Type II PS+: 0.523±0.033). PS- sickle RBC had a uniformly low PLSCR activity similar to normal RBC (NBD-PC internalization fractions ∼ 0.1). In mature sickle RBC, PLSCR was more active in PS+ cells (PS-: 0.097±0.096; Type I PS+: 0.163±0.070, Type II PS+: 0.248±0.043; n=3; PS- vs Type I PS+: p=0.06; PS- vs Type II PS+: p=0.04; Type I versus Type II: p=0.03). TfR+ reticulocytes had increased APLT and PLSCR activity compared to mature sickle RBC, but there was no apparent relationship between PLSCR and external PS. Since dense sickle RBC had markedly inhibited APLT, we evaluated the relationship between dehydration and APLT activity. Dehydration of AA RBC from an MCHC of 35.6±2.2 to 49.2±2.0 g/dL inhibited APLT (from 0.484±0.068 to 0.301±0.076; n=7, p= 0.01). Dehydration of SS RBC from an MCHC of 34.8±3.5 to 50.1±3.9 g/dL also inhibited APLT (from 0.460±0.060 to 0.361±0.047; n=3, p=0.006), but not as low as in SS RBC dehydrated in vivo (0.222±0.036 at 44.7±5.6 g/dL; n=4, p=0.007 vs. SS RBC dehydrated in vitro). Rehydration of AA and SS RBC that had been dehydrated in vitro reversed APLT inhibition. However, APLT activity was not reversed upon rehydration of sickle RBC dehydrated in vivo. In summary, our data show that:1.many dense sickle RBC with significantly inhibited APLT are PS-, indicating that APLT inhibition alone does not result in PS externalization;2.dehydration contributes to, but is not entirely responsible for, the APLT inhibition seen in dense sickle RBC; and3.PS+ sickle RBC have increased PLSCR activity.

Enhanced Phosphatidylserine Scrambling in Oxidatively Challenged Red Blood Cells.

Red blood cells (RBC) that abnormally expose phosphatidylserine (PS) contribute to the pathophysiology of several hemoglobinopathies. PS exposure requires inactivation of the flippase that transports PS from the outer to the inner membrane monolayer, and activation of a phospholipid scrambling process. To evaluate the role of increased oxidative stress in this process, we compared RBC from transgenic sickle mice (Berkeley type) with RBC from peroxiredoxin 2 (prdx) knock-out mice (prdx −/−). These mice lack one of the most prevalent cytosolic antioxidant molecules. This molecule, also known as calpromotin, was previously implicated in membrane abnormalities of the dense sickle RBC population. Mice that lack prdx are slightly anemic, have reduced RBC survival and exhibit a subpopulation of older highly oxidized RBC. The prdx −/− strain did not show a subpopulation of PS-exposing cells in freshly collected blood and the flippase activity, measured by transbilayer kinetics of the fluorescent probe NBD-PS, was normal. In contrast, blood collected from the sickle mice showed a large subpopulation with decreased flippase activity and exhibited a subpopulation of PS-exposing RBC that lack flippase activity. Flippase inhibition induced with vanadate or NEM did not increase the PS exposure of prdx −/− RBC incubated with high levels of Ca2+, indicating that there was no increased Ca2+ influx. In sickle cells, elevated intracellular Ca2+ was evident under similar loading conditions. Loading RBC with 0.1 mM Ca2+, but not lower concentrations, using Ca-ionophore resulted in bilayer scrambling and PS exposure in both strains as well as in normal control mice. The rate of PS scrambling was increased 1.5-fold in sickle mice compared to normal mouse RBC. While the scrambling rate was normal in the young, not oxidized prdx−/− RBC, it was increased 3-fold in the older highly oxidized prdx −/− RBC as compared to normal mouse RBC. The sulfhydryl modifiers NEM or PDA caused flippase inhibition, and altered the PS scrambling rate in normal mouse RBC as reported earlier. Both sickle cells and the older oxidized prdx −/− RBC showed a reduced susceptibility to NEM and PDA, while the younger prdx −/− RBC exhibited a normal sensitivity to these compounds. This suggests that both prdx −/− RBC and sickle cells have sustained similar sulfhydryl damage leading to enhanced scrambling. Exposure to three well-known oxidants (0.1–0.5 mM cumene hydroperoxide, tert-butyl hydroperoxide or hydrogen peroxide) did not increase the percentage of oxidized cells or PS exposure in prdx −/− RBC compared to normal RBC. This indicates that targeted sulfhydryl modification but not general short-term oxidative stress impacts the loss of phospholipid asymmetry. These data confirm that increased oxidative sulfhydryl damage results in a higher propensity for phospholipid scrambling. The presence of active prdx is important to maintain PS asymmetry as it prevents accelerated phospholipid scrambling. In those cells in which the flippase is also inactivated, PS exposure becomes apparent. The loss of flippase activity is much more prevalent in sickle RBC, indicating that prdx does not play an important role in protecting the flippase from inactivation. It can be expected that PS-exposing cells are rapidly removed from the circulation, as they resemble apoptotic cells, which may explain their absence in blood from prdx−/− mice. The presence of PS-exposing RBC in the circulation of sickle mice suggests that the formation of these cells overwhelms their removal.

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

Failures in Clinical Treatment of Staphylococcus aureus Infection with Daptomycin Are Associated with Alterations in Surface Charge, Membrane Phospholipid Asymmetry, and Drug Binding

Increasingly frequent reports have described the in vivo loss of daptomycin susceptibility in association with clinical treatment failures. The mechanism(s) of daptomycin resistance is not well understood. We studied an isogenic set of Staphylococcus aureus isolates from the bloodstream of a daptomycin-treated patient with recalcitrant endocarditis in which serial strains exhibited decreasing susceptibility to daptomycin. Since daptomycin is a membrane-targeting lipopeptide, we compared a number of membrane parameters in the initial blood isolate (parental) with those in subsequent daptomycin-resistant strains obtained during treatment. In comparison to the parental strain, resistant isolates demonstrated (i) enhanced membrane fluidity, (ii) increased translocation of the positively charged phospholipid lysyl-phosphotidylglycerol to the outer membrane leaflet, (iii) increased net positive surface charge (P < 0.05 versus the parental strain), (iv) reduced susceptibility to daptomycin-induced depolarization, permeabilization, and autolysis (P < 0.05 versus the parental strain), (v) significantly lower surface binding of daptomycin (P < 0.05 versus the parental strain), and (vi) increased cross-resistance to the cationic antimicrobial host defense peptides human neutrophil peptide 1 (hNP-1) and thrombin-induced platelet microbicidal protein 1 (tPMP-1). These data link distinct changes in membrane structure and function with in vivo development of daptomycin resistance in S. aureus. Moreover, the cross-resistance to hNP-1 and tPMP-1 may also impact the capacity of these daptomycin-resistant organisms to be cleared from sites of infection, particularly endovascular foci.

Purpurin-18 in Combination with Light Leads to Apoptosis or Necrosis in HL60 Leukemia Cells¶

Photodynamic therapy (PDT), a cancer treatment using a photosensitizer and visible light, has been shown to induce apoptosis or necrosis. We report here that Purpurin-18 (Pu18) in combination with light induces rapid apoptotic cell death in the human leukemia cell line (HL60) at low doses and necrosis at higher concentrations. Cells treated with Pu18 and light under apoptotic conditions exhibited DNA laddering and an increase in both cellular content of subdiploid DNA and externalization of phosphatidylserine (PS), indicating DNA fragmentation and loss of membrane phospholipid asymmetry. In the absence of light activation, Pu18 at nanomolar concentrations had no detectable cytotoxic effect. Caspase-3 activity was increased even after 1 h from treatment with low doses of Pu18 and light. The PS exposure and nuclear features of apoptosis were prevented by treatment of cells before illumination with caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK). Conversely, the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) failed to suppress the apoptosis. No protective effect of the three caspase inhibitors was observed when the cells were exposed to necrotic concentrations of Pu18 and light. Our results show that caspase-3, but not caspase-1, is involved in the signaling of apoptotic events in PDT with Pu18-induced apoptosis of HL60 cells. Moreover, both the time course of PS exposure and the effect of caspase inhibitors on it indicate that it is regulated in the same manner as DNA fragmentation.

Phospholipid scramblases: An overview

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca 2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.

In vitro susceptibility of Staphylococcus aureus to thrombin-induced platelet microbicidal protein-1 (tPMP-1) is influenced by cell membrane phospholipid composition and asymmetry

Thrombin-induced platelet microbicidal proteins (e.g. tPMP-1) are small cationic peptides released from mammalian platelets. As the cytoplasmic membrane (CM) is a primary target of tPMPs, distinct CM characteristics are likely to affect the cells' susceptibility profiles. In Staphylococcus aureus, CM surface charge and hydrophobicity are principally determined by the content and distribution of its three major phospholipid (PL) constituents: negatively charged phosphatidylglycerol (PG) and cardiolipin (CL) and positively charged lysyl-PG (LPG). PL composition profiles, and inner vs outer CM leaflet PL distributions, were compared in an isogenic tPMP-susceptible (tPMP(S)) and -resistant (tPMP(R)) S. aureus strain pair (ISP479C vs ISP479R respectively). All PLs were asymmetrically distributed between the outer and inner CM leaflets in both strains. However, in ISP479R, the outer CM leaflet content of LPG was significantly increased vs ISP479C (27.3+/-11.0 % vs 18.6+/-7.0 % respectively; P=0.05). This observation correlated with reduced binding of the cationic proteins cytochrome c, poly-L-lysine, tPMP-1 and the tPMP-1-mimetic peptide, RP1, to tPMP-1(R) whole cells and to model liposomal CMs with LPG content and distribution similar to that of tPMP-1(R) strains. Collectively, selected CM parameters correlated with reduced staphylocidal capacities of tPMP-1 against certain S. aureus strains, including relative increases in outer CM leaflet positive charge and reduced surface binding of cationic molecules. These findings offer new insights into mechanisms of antimicrobial peptide susceptibility and resistance in S. aureus.

Investigation on the early events of apoptosis in senescent erythrocytes with special emphasis on intracellular free calcium and loss of phospholipid asymmetry in chronic renal failure

Background The pathophysiological link between increased blood concentrations of factors responsible for the derangement and erythrocyte membrane functions in chronic renal failure (CRF) patients are not thoroughly elucidated. We studied the erythrocyte characteristics and phospholipid asymmetry loss in CRF patients with different grades of uremia and also examined the involvement of intracellular free Ca 2+ in early events of apoptosis in uremic erythrocytes. Methods The studied population consisted of 90, age and sex matched control subjects (Group I) and 238 CRF cases divided into 3 groups (Group II, III and IV) according to urea concentrations and complexity of secondary complications. Erythrocyte membrane fluidity determined by binding of MC540. Intracellular free Ca 2+ concentration was determined by the 2-wavelength method by using fluorescent calcium-sensitive probe FURA-2AM. Measurement of erythrocyte phosphatidylserine exposure by flow cytometry using Annexin V-FITC. Results Cholesterol shedding increased with increasing severity of uremic complications. Erythrocytes from Group II show mild echinocyte or formation of spicules on the erythrocyte membrane surface whereas in Group III and IV they were echinocytic. Binding of MC540 was significantly higher with progression of uremic complications. Surface charge of uremic erythrocyte membrane was significantly reduced when compared with control subjects. Intracellular free Ca 2+ was positively correlated with binding of MC540 and surface hydrophobicity. The phosphatidylserine exposure of erythrocytes was significantly higher ( p < 0.001) in uremic patients when compared with controls. Conclusions Phosphatidylserine (PS) exposure erythrocytes were significantly increased in uremic patients when compared with controls. Uremic complications predisposes to membrane damages in erythrocytes.

Macrophage-mediated Bystander Effect Triggered by Tumor Cell Apoptosis

Restoration of apoptosis is an important therapeutic strategy for cancer, but bystander effects may be crucial to treatment success. We examined the involvement of bystander effects in the outcome of pro-apoptotic treatments and investigated the role of macrophages. Using a murine N202 breast cancer chamber model and intravital microscopy, we observed bystander apoptosis in vivo in mixed spheroids consisting of bystander N202 cells plus modified N202 cells overexpressing the p14ARF N-terminal region, which promotes p53-mediated apoptosis. The effect was not observed in cocultures in vitro, and could not be transferred through conditioned medium from modified N202 cells. However, if macrophages were also included in the N202 co-cultures, bystander apoptosis was restored, and correlated with elevated surface expression of phosphatidyl serine, a macrophage recognition molecule, on the modified N202 cells. Bystander killing was not observed in cocultures of N202 cells plus macrophages plus cisplatin-treated or 5-fluorouracil-treated N202 cells, where apoptosis induction in the target cell population was inefficient, suggesting that specific activation of macrophages by apoptotic tumor cells was required. The results suggest that pro-apoptotic therapies benefit both from the intrinsic vulnerability of cancer cells to apoptosis and from an innate immune response that amplifies the therapeutic effect.

Ethyl acetate extract of Chinese medicinal herb Sarcandra glabra induces growth inhibition on human leukemic HL-60 cells, associated with cell cycle arrest and up-regulation of pro-apoptotic Bax/Bcl-2 ratio

Sarcandra glabra (Thunb.) Nakai, colloquially known as Caoshanhu, is a Chinese medicinal herb with reported anti-tumor, anti-inflammatory, anti-viral and non-specific immunoenhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are largely unknown and its mode of action has never been investigated. In this study, the anti-tumor property of ethyl acetate (EA) extract of S. glabra was investigated by determining its in vitro growth-inhibitory effects on a panel of human cancer cell lines of different histotypes. Growth inhibition of the EA extract on the cancer cells seemed to be selective, and the leukemic HL-60 was found to be the most responsive after 48 h of treatment (IC50=58 microg/ml). Flow cytometric studies further illustrated that the extract might interfere with DNA replication and thus arrested the cell cycle at S phase in the leukemic cells, followed by DNA fragmentation and loss of phospholipid asymmetry in the plasma membrane after 72 h of treatment. Concurrently, the pro-apoptotic Bax/Bcl-2 ratio was also up-regulated by more than 178% of the control level. All these findings suggested that the extract had initiated apoptosis to kill the leukemic cells. Results from this pioneer study help to establish a scientific foundation for future research and development of the bioactive ingredients in EA extract of S. glabra as efficacious anti-cancer agents.

Membrane Lipid Alterations in Hemoglobinopathies

The red blood cell (RBC) membrane is a complex mixture of lipids and proteins. Hundreds of phospholipid molecular species spontaneously arrange themselves in a lipid bilayer and move rapidly in the plane as well as across the bilayer in a dynamic but highly organized fashion. Areas enriched in certain lipids determine proper protein function. Phospholipids are asymmetrically distributed across the lipid bilayer with phosphatidylserine (PS) exclusively on the inside. Both the composition and organization of the RBC membrane is well maintained. Alterations lead to apoptosis during erythropoiesis or early demise of the cell in the circulation. The mechanisms that govern the maintenance of the lipid bilayer are only recently being unraveled at the individual protein level. Oxidized lipids are rapidly repaired using fatty acids taken up from plasma to maintain membrane integrity. Several isoforms of a RBC acyl-Coenzyme A (CoA) synthase have been reported, as well as the first member of a family of lysophospholipid acylCoA acyltransferases. Phospholipid asymmetry is maintained by the recently identified RBC amino-phospholipid translocase. These enzymes, essential in maintaining membrane lipid organization, are affected by oxidant stress or an increase in cytosolic calcium. Normal lipid composition and organization is lost in subpopulations of RBC in hemoglobinopathies such as sickle cell disease and thalassemia. Despite elaborate antioxidant systems, lipids and membrane proteins, including those that maintain lipid organization, are damaged in these cells. This in turn leads to improper repair of damaged RBC membranes and altered interactions of RBCs with other blood cells and plasma components that play a role in the pathology that defines these disorders. The altered lipid bilayer in RBCs in hemoglobinopathies leads to premature removal (anemia) and imbalance in hemostasis, and plays a role in vaso-occlusive crisis in sickle cell disease. Lipid breakdown products of PS-exposing cells result in vascular dysfunction, including acute chest syndrome in sickle cell disease. In summary, altered membrane lipids play an important role in the pathology of hemoglobinopathies and characterization of the proteins involved in lipid turnover will elucidate the pathways that maintain plasma membrane organization and cellular viability.

The Aspergillus fumigatus metacaspases CasA and CasB facilitate growth under conditions of endoplasmic reticulum stress

We have examined the contribution of metacaspases to the growth and stress response of the opportunistic human mould pathogen, Aspergillus fumigatus, based on increasing evidence implicating the yeast metacaspase Yca1p in apoptotic-like programmed cell death. Single metacaspase-deficient mutants were constructed by targeted disruption of each of the two metacaspase genes in A. fumigatus, casA and casB, and a metacaspase-deficient mutant, DeltacasA/DeltacasB, was constructed by disrupting both genes. Stationary phase cultures of wild-type A. fumigatus were associated with the appearance of typical markers of apoptosis, including elevated proteolytic activity against caspase substrates, phosphatidylserine exposure on the outer leaflet of the membrane, and loss of viability. By contrast, phosphatidylserine exposure was not observed in stationary phase cultures of the DeltacasA/DeltacasB mutant, although caspase activity and viability was indistinguishable from wild type. The mutant retained wild-type virulence and showed no difference in sensitivity to a range of pro-apoptotic stimuli that have been reported to initiate yeast apoptosis. However, the DeltacasA/DeltacasB mutant showed a growth detriment in the presence of agents that disrupt endoplasmic reticulum homeostasis. These findings demonstrate that metacaspase activity in A. fumigatus contributes to the apoptotic-like loss of membrane phospholipid asymmetry at stationary phase, and suggest that CasA and CasB have functions that support growth under conditions of endoplasmic reticulum stress.

Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene

The P-type Mg 2+-ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (− 318/+ 193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (− 1026/+ 193). Serial 5′ deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells.

Flippase Activity Decreases throughout Erythrocyte Life-Span in Normal and Sickle Mice.

Phosphatidylserine (PS) exposure contributes to recognition and removal of aged erythrocytes. PS exposure can only arise in cells in which the aminophospholipid translocase, or flippase, is defunct, or otherwise PS would be transported back into the inner cell monolayer. To provide evidence that flippase activity is reduced in old erythrocytes, we studied mouse erythrocytes that were biotinylated in vivo. This way we were able to distinguish the aging, biotinylated, RBC population from the newly formed, unbiotinylated RBC. Detection of the biotinylation with fluorescent streptavidin was combined with a flow cytometric assay for flippase activity using NBD-PS. We found that flippase activity decreased over time during the life-span of the RBC, as shown by a decrease in flippase activity of the biotinylated RBC compared to the newly formed RBC. A particularly high flippase activity was found in CD71-positive reticulocytes. Murine sickle cells show a much higher erythrocyte turnover than normal erythrocytes, with a 25–50% newly formed population each day and complete elimination of all biotinylated cells in 10 days. As in control mice, flippase activity decreased during the (short) life-span of sickle RBC and at all times, a subpopulation of sickle cells had a markedly reduced flippase activity. High flippase activity was only found in newly released erythrocytes. Our data indicate that the flippase activity decreases throughout the life-span of erythrocytes and can be markedly different in subpopulations of RBC in vivo. This decrease reduces the cell's potential to maintain its phospholipid asymmetry, which results in PS exposure as a removal signal for the damaged cell. We conclude that the inability of sickle RBC to maintain flippase activity is related to the decreased survival time of those cells.