Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique

Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system.     

Antimycin A-induced apoptosis of HL-60 cells

BACKGROUND: Previous experiments in our laboratory investigating apoptosis induced in HL-60 cells by camptothecin (CAM) have revealed that the sequence and rapidity of the apoptotic phenomena in an individual cell depend on the proliferative state of that cell when it encounters CAM. The role of mitochondria in HL-60 apoptosis was explored using an inhibitor of oxidative phosphorylation, antimycin A (AMA). METHODS: Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC), uptake of propidium iodide (PI), and DNA content after membrane fixation/permeabilization were monitored by flow cytometry. Z-VAD-FMK was used to inhibit caspases. Fluorescence microscopy was used to examine cell morphology. RESULTS: Cells in the G1 phase of the cell cycle were the first to exhibit signs of apoptosis in response to 100 microM AMA and some of these cells disintegrated without exposing to phosphatidylserine (PS). Caspase inhibition prevented fragmentation of DNA, the nucleus, and the cell, but only delayed PS exposure and loss of plasma membrane integrity. CONCLUSIONS: The highly active mitochondria of G1-phase HL-60 cells make them particularly sensitive to AMA. PS exposure and plasma membrane damage are mediated by noncaspase molecules released from mitochondria. We hypothesize that if mitochondria are subjected to a sufficiently severe insult, whether indirectly as a result of extensive CAM-induced DNA damage or directly by the effect of AMA on electron transport, the nature and quantities of the proapoptotic molecules released are such that apoptosis proceeds to the point of cell disintegration before the PS exposure pathway is complete.     

Relationship between actin microfilaments and plasma membrane changes during apoptosis of neoplastic cell lines in different culture conditions

In this study we investigated the relationship between the reorganisation of actin cytoskeleton and the changes at cell surface level (i.e. PS exposure and blebbing) in two neoplastic cell lines during apoptosis: Chang liver cells (adherent culture) and promyelocytic HL-60 cells (suspension culture), treated with the podophyllotoxin derivative VP16. The morphological analysis, performed by means of conventional fluorescence microscopy and confocal laser scanning microscopy, on Chang cells showed that onset and progress of the two processes are synchronised. The initial disassembly of stress fibers was associated with the early PS exposure on the cell surface. Moreover, the accumulation of actin at cortical level appeared strongly associated with an intense labelling for Annexin V and, in some cases, especially in the areas of membrane blebbing. The double staining for actin and PS exposure, quantitatively analysed by flow cytometry in HL-60 cells after different treatment times, demonstrated that the decrease of Annexin V binding in the late stages of apoptosis is associated with the strong reduction of actin labelling probably also due to a proteolytic cleavage. These events were also partially related to variations of the functional state of mitochondria, by analysing cytofluorometrically the dissipation of the inner membrane potential (delta psi m).     

Preparation and characterization of immunoconjugates for antibody-targeted photolysis.

Monoclonal antibody (MAb)-dextran-tin(IV) chlorin e6 (SnCe6) immunoconjugates were prepared by a new technique involving the use of reducing, terminal-modified dextran carriers and site-specific modification of the Fc oligosaccharide moiety on the antibodies. Dextran carriers were synthesized to increase the number of SnCe6 molecules attached to a MAb. The dextran carriers were coupled to the MAb via a single, chain-terminal hydrazide group to prevent aggregation of MAbs. Conjugates were prepared with antimelanoma MAb 2.1 containing up to 18.9 SnCe6 molecules per MAb. Under neutral conditions, no hydrolysis of the hydrazone bond between the MAb and the dextran carrier could be detected, and the hydrazone was not stabilized by reduction with NaCNBH3 or NaBH4. Analysis of the purified immunoconjugates showed that approximately two dextran carrier chains were attached to a MAb regardless of the number of SnCe6 molecules linked to a dextran carrier. Site-specific covalent attachment of the SnCe6-dextran chains to the MAb was confirmed by SDS-PAGE. HPLC analysis of the conjugates gave a single species eluting in the range of 200-240 kDa. As determined by a competitive inhibition radioimmunoassay using viable SK-MEL-2 human malignant melanoma cells, the conjugates showed excellent retention of antigen-binding activity relative to unconjugated MAb.     

Photodynamic activities of sulfonamide derivatives of porphycene on nasopharyngeal carcinoma cells

Two sulfonamide derivatives of porphycene, namely PS6 and PS6A, were synthesized, and their photodynamic efficacies on the nasopharyngeal carcinoma (NPC) cell line NPC/CNE-2 were evaluated. By comparing the 50% lethal concentrations (LC(50)) of these photosensitizers, we found that PS6A with a cationic ammonium group on the side chain exhibited potent photocytotoxicity on the NPC cell line. At a light dose of 1 J/cm(2), the LC(50) values of PS6 and PS6A for NPC cells were 11.6 and 1.92 microM, respectively. CNE-2 was found to rapidly take up PS6A in the first hour of incubation, and the uptake kinetics steadily increased to a plateau level after 18 h of incubation. The uptake of PS6A was temperature dependent. Over 99% of CNE-2 cells were sensitized by PS6A 24 h after drug treatment. Collapse of the mitochondrial membrane potential was also observed in PS6A photodynamic therapy (PDT)-treated CNE-2 cells 1.5 h after PDT. Confocal microscopy revealed that PS6A was predominantly localized in the mitochondria, lysosomes and Golgi bodies of NPC cells. Significant genotoxicity was not observed in CNE-2 cells. In functional studies, the in vitro formation of a capillary-like network of human umbilical vein endothelial cells in Matrigel was greatly inhibited by PS6A PDT in a dose-dependent manner. In conclusion, PS6A mediates both in vitro antitumor and antiangiogenic activities. PS6A might be a candidate for photodynamic treatment of NPCs.     

Correlation of cell surface alterations with contractile response in laser microbeam irradiated myocardial cells : A scanning electron microscope study

The contractile behavior and surface morphology of cultured neonatal rat heart cells were examined by phase contrast and scanning electron microscopy (SEM) following laser irradiation of single mitochondria. Irradiation always resulted in damage to the target mitochondrion (as determined by phase microscopy) and was associated with one of three contractile states, each of which correlated with a specific surface morphology over the irradiated mitochondrion. The results demonstrate that: (1) changes in the contractile activity of the cell correlate directly with morphological changes in the target organelle and in the membrane overlying the target organelle; (2) when the contractile activity of the cell remains unchanged, the morphology of the membrane overlying the target organelle appears normal via SEM even though the organelle is visibly damaged as judged by phase contrast microscopy; (3) the correlation between contractile behavior and surface morphology was the same regardless of which cell surface the laser beam passed through when entering the cell (i.e., through the cell surface directly apposed to the glass or through the free cell surface directly exposed to the medium); (4) the mitochondrial lesions could be compared to lesions made in dried red blood cells irradiated from either surface. (Again the lesions appeared identical regardless of the cell surface through which the laser beam entered.) These observations suggest that laser damage is produced equally in all directions from the focal point.     

Antimicrobial peptide magainin 2-induced rupture of single giant unilamellar vesicles comprising E. coli polar lipids

Most antimicrobial peptides (AMPs) damage the cell membrane of bacterial cells and induce rapid leakage of the internal cell contents, which is a main cause of their bactericidal activity. One of the AMPs, magainin 2 (Mag), forms nanopores in giant unilamellar vesicles (GUVs) comprising phosphatidylcholine (PC) and phosphatidylglycerol (PG), inducing leakage of fluorescent probes. In this study, to elucidate the Mag-induced pore formation in lipid bilayer region in E. coli cell membrane, we examined the interaction of Mag with single GUVs comprising E. coli polar lipids (E. coli-lipid-GUVs). First, we investigated the Mag-induced leakage of a fluorescent probe AF488 from single E. coli-lipid-GUVs, and found that Mag caused rupture of GUVs, inducing rapid AF488 leakage. The rate constant of Mag-induced GUV rupture increased with the Mag concentration. Using fluorescence microscopy with a time resolution of 5 ms, we revealed the GUV rupture process: first, a small micropore was observed in the GUV membrane, then the pore radius increased within 50 ms without changing the GUV diameter, the thickness of the membrane at the pore rim concomitantly increased, and eventually membrane aggregates were formed. Mag bound to only the outer monolayer of the GUV before GUV rupture, which increased the area of the GUV bilayer. We also examined the physical properties of E. coli-lipid-GUVs themselves. We found that the rate constant of the constant tension-induced rupture of E. coli-lipid-GUVs was higher than that of PG/PC-GUVs. Based on these results, we discussed the Mag-induced rupture of E. coli-lipid-GUVs and its mechanism.     

Studies on the induction and expression of T cell-mediated immunity. VIII. Effector-target junctions and target cell membrane disruption during cytolysis.

Target cell destruction following contact of the target cell by specific alloimmune cytotoxic thymus-derived lymphocytes (CTL) has been examined by time-lapse film (TLF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Effector-target conjugates of murine CTL with leukemia cells were prepared for use in these studies. TLF shows that contact of the two cells results in tumor cell zeosis involving violent membrane blebbing, and subsequent tumor cell death. TEM of the contact region shows that the CTL-tumor cell junction is extremely adherent. Examination of conjugates incubated at 37 °C to permit tumor cell lysis shows tumor cell membrane stretching and rupture, and tumor cell membrane fragments adhering to CTL. Close examination of the contact region has revealed electron-lucent junctions spanning the gap between the two cell membranes, but no packaging or secretory apparatus was prominent. The results are consistent with the mechanism of cell-mediated cytolysis being a membrane phenomenon involving junctions connecting the CTL and target cell and the initial target cell lesion observable as a stretching and rupture. The shear force of vigorous cell movements is most likely responsible for this target membrane tearing, creating a target cell lesion which results in loss of osmotic integrity and cell death.     

Laser scanning confocal microscopy of the hearing organ: fluorochrome-dependent cellular damage is seen after overexposure

In order to combine laser confocal microscopy with physiological measurements, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was explored on isolated coils from the cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.     

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

Comparative studies on cytopathic effects induced by vesicular stomatitis virus in two cell types.

Infection of mouse L cells with vesicular stomatitis virus (VSV) leads to an extensive cell fusion, while porcine kidney stable (PS) cells infected with VSV show only cell rounding. Therefore, comparative morphological studies on the infection of the two cell lines were carried out using a transmission or scanning electron microscope and an immunofluorescence microscope. PS cells infected with VSV contrasted to L cells infected with the same virus in the following two points; (1) the principal site of VSV maturation was the intracytoplasmic vacuolar membrane in PS cells and the plasma membrane in L cells. However, it was found that viral glycoprotein was present on the cell surface of infected PS cells; (2) the morphological changes at the cell surface of infected PS cells occurred much earlier and were severer than those at the cell surface of infected L cells. From these observations, we discuss the possibility that the surfaceembrane of PS cells is too sensitive to the VSV-induced cell damage to cause cell fusion.     

Antinematodal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Caenorhabditis elegans.

The antinematodal activity and mechanism of a 23-mer antimicrobial peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed a strong antinematodal activity against the eggs and worms of Caenorhabditis elegans. To investigate the antinematodal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. C. elegans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide (PI) staining than normal cells. Confocal microscopy showed that the peptide was localized in the egg's shell and cell membrane. The action of the peptide against C. elegans membranes was examined by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w). The result suggests that PMAP-23 may exert its antinematodal activity by disrupting the structure of the cell membrane via pore formation or via direct interaction with the lipid bilayers.     

Inner Structure of Intact Chloroplasts Observed by a Low Temperature Laser Scanning Microscope

Inner structure of isolated intact chloroplasts was observed for the first time by a method of laser scanning microscopy at the temperature of liquid nitrogen at 77 K. The microscope, based on gradient index optics, has a maximum resolution of 440 nm at the wavelength of 650 nm. Chloroplasts were excited into the Q-band of chlorophyll b by a krypton laser line at 647.6 nm and fluorescence was detected using two different interference filters. The 680 nm interference filter detects the regions where photosystem (PS) 2 mainly occurs, the 730 nm interference filter detects domains with predominant location of PS1. Since PS1 occurs mainly in stroma lamellae, whereas PS2 occurs mainly in grana regions we were able to view the structure of thylakoid membrane in isolated intact chloroplast that is the closest to in vivo state.     

Vesicles mimicking normal and cancer cell membranes exhibit differential responses to the cell-penetrating peptide Pep-1

The cell-penetrating peptide (CPP) Pep-1 presents a great potential in drug delivery due to its intrinsic property to cross plasma membrane. However, its mechanism of entry into the cell remains unresolved. In this study, we compare the selectivity of Pep-1 towards vesicles mimicking normal and cancer cell membranes. The interaction was performed in a wide range of peptide-to-lipid molar ratios using infrared (IR), fluorescence, scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. At low peptide concentration, fluorescence experiments show that lipid-phosphatidylserine (PS) seems to enable Pep-1 translocation into cancer cell membrane as evidenced by the blue shift of its maximal emission wavelength. DSC data show that Pep-1 induces segregation of lipids. At high peptide concentration, IR data indicate that the interaction of Pep-1 is relatively stronger with normal cell membrane than with cancer cell membrane through the phosphate groups, while the interaction is weaker with normal cell membrane than with cancer cell membrane through the carbonyl groups. TGA and DSC data reveal that vesicles of normal cell membrane are thermally more stable than vesicles of cancer cell membrane. This suggests that the additional lipid PS included in cancer cell membrane has a destabilizing effect on the membrane structure. SEM images reveal that Pep-1 form superstructures including spherical particles and fibrils in the presence of both model membranes. PS seems to enhance peptide transport across cellular membranes. The biophysical techniques in this study provide valuable insights into the properties of CPPs in drug delivery systems.     

Reactive effect of low intensity He-Ne laser upon damaged ultrastructure of human erythrocyte membrane in Fenton system by atomic force microscopy

To find out the mechanism of modulating the deformability of erythrocytes with low intensity He-Ne laser action, we studied the effect of low intensity He-Ne laser on the ultrastructure of human erythrocyte membrane. Erythrocytes were treated with free radicals from a Fenton reaction system before exposing them to low intensity He-Ne laser. The ultrastructure of damaged erythrocyte membrane was examined by atomic force microscopy. The results showed that the erythrocyte membrane became very rough and the molecules on the surface of the membrane congregated into particles of different magnitudes sizes after treating with free radicals. Comparing the degree of congregation of the molecular particles in the non-irradiated group and the He-Ne laser irradiated （9 mW and 18 mW） group, we found the average size of molecular particles in the laser irradiated group was smaller than that in the non-irradiated group, indicating that the low intensity laser had repairing function to the damage of erythrocyte membrane produced by the free radicals.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (～ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Bovine Lactadherin as a Calcium-independent Imaging Agent of Phosphatidylserine Expressed on the Surface of Apoptotic HeLa Cells

Bovine lactadherin holds a stereo-specific affinity for phosphatidylserine (PS) membrane domains and binds at PS concentrations lower than the benchmark PS probe, annexin V. Accordingly, lactadherin has recognized PS exposure on preapoptotic immortalized leukemia cells at an earlier time point than has annexin V. In the present study, the cervical cancer cell line HeLa has been employed as a model system to compare the topographic distribution of PS with the two PS binding proteins as adherent cells enter the apoptotic program. HeLa cells were cultured on glass-bottom Petri dishes, and apoptosis was induced by staurosporine. Fluorescence-labeled lactadherin and/or annexin V were used to detect PS exposure by confocal microscopy. Both lactadherin and annexin V staining revealed PS localized to plasma membrane rim and blebs. In addition, lactadherin identified PS exposure on long filopodia-like extensions, whereas annexin V internalized in granule-like structures. All in all, the data further delineate the differences in PS binding patterns of lactadherin and annexin V. (J Histochem Cytochem 57:907–914, 2009)     

Hypochlorous acid-derived modification of phospholipids: characterization of aminophospholipids as regulatory molecules for lipid peroxidation

Hypochlorous acid (HOCl), an inflammatory oxidant derived from neutrophil myeloperoxidase, can chlorinate cytosolic proteins and nuclear DNA bases of target cells by passing through the cell membrane. However, little is known about the consequences of HOCl-derived modification of cell membrane components, including phospholipids. In this study, we characterize the reaction of HOCl with phospholipid molecules and found that aminophospholipids are the key molecules that chemically regulate lipid peroxidation. Upon incubation with HOCl, the peroxidation of egg yolk phosphatidylcholine was significantly enhanced in the presence of phosphatidylethanolamine (PE). In contrast, the peroxidation was significantly inhibited in the presence of phosphatidylserine (PS). On the basis of mass spectrometric and electron paramagnetic resonance characterization, the initiator of the peroxidation was identified as the nitrogen-centered radical originating from PE-derived chloramines, especially N,N-dichlorinated PE, a major product in the HOCl-modified PE. Although PS was also chlorinated upon reaction with HOCl, the formed chloramine rapidly decomposed to phosphatidylglycolaldehyde, a novel class of lipid aldehyde. Formation of phosphatidylglycolaldehyde was also confirmed in the porcine brain PS and erythrocyte cell membrane ghost exposed to HOCl. These results provide a novel mechanism for the HOCl-induced oxidative damage and its endogenous protection in the cell membrane at the site of inflammation.     

Measurement of DNA damage associated with apoptosis by laser scanning cytometry.

BACKGROUND: Phosphatidylserine (PS) binding by annexin V (AV) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV(+) cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623-8, 2000). METHODS: The "halo" assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. RESULTS: AV(+) cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV(+) propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are downstream from caspase activation. CONCLUSIONS: This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.