Electric field-directed cell shape changes, displacement, and cytoskeletal reorganization are calcium dependent.

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field-induced disorganization of the stress fiber pattern, which was reduced when stimulation was conducted in calcium-depleted buffer or in buffer containing calcium antagonist CoCl2, calcium channel blocker D-600, or calmodulin antagonist trifluoperazine. Treatment with calcium ionophore A23187 had similar effects, except that the presence of D-600 did not reduce the stress fiber disruption. The calcium-sensitive photoprotein aequorin was used to monitor changes in intracellular-free calcium. Electric stimulation caused an increase of calcium to the micromolar range. This increase was inhibited by calcium-depleted buffer or by CoCl2, and was reduced by D-600. A calcium-dependent mechanism is proposed to explain the observed field-directed cell shape changes, preferential orientation, and displacement.

Effects of cholesterol on lipid organization in human erythrocyte membrane.

The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.

Direct observation of domains in wet lipid bilayers.

Domain structure and phase separation in hydrated lipid bilayers have been imaged directly by selected reflection dark-field electron microscopy. Domains in multicomponent bilayers are much smaller than those in single component bilayers, in agreement with results obtained by selected area electron diffraction.

Electron diffraction of wet biological membranes.

Electron diffraction patterns were obtained for the first time from single wet phospholipid bilayers and from wet human erythrocyte membranes by using a temperature-controlled electron microscope hydration stage. Selective area diffraction showed the existence of semicrystalline domains. A structural transition was observed at the transition temperature of the wet dipalmitoyl lecithin bilayer.

Membrane fusion through point defects in bilayers.

Fusion between bilayers of mixed egg phosphatidylcholine and soybean phosphatidylethanolamine was induced by freezing and thawing. Contact points between bilayers were observed by freeze fracture electron microscopy, and isotropic molecular motional averaging was detected by phosphorus-31 nuclear magnetic resonance under fusion conditions. A molecular model of point defect structure is proposed as an intermediate stage of fusion.

Fluorinated molecule as a tracer: difluoroserotonin in human platelets mapped by electron energy-loss spectroscopy.

The intracellular distribution of fluorine has been delineated in human platelets incubated with 4,6-difluoroserotonin, utilizing a scanning-transmission electron microscope equipped with an energy-loss spectrometer. Discrete intracellular structures corresponding in location to dense bodies contained high concentrations of fluorine. Electron energy-loss spectroscopy, which apparently can detect less than 10(-20) gram of fluorine in an area of 10 square nonometers, can thus localize fluorinated tracer molecules with biological activity.

Acute fatal sarcocystosis hepatitis in an Indo-Pacific bottlenose dolphin (Tursiops aduncus) in Hong Kong.

Unlike most species in the genus Sarcocystis, Sarcocystis canis has a broad intermediate host range. Its life cycle is incompletely known and most reports are from the USA. Here we report fatal hepatitis in a 4year old male Indo-Pacific bottlenose dolphin (Tursiops aduncus) from Hong Kong associated with a S. canis-like infection. Diagnosis was made based on clinical presentation, histopathology, transmission electron microscopy (TEM), and molecular characterization. Microscopically, S. canis-like like infection was confined to the liver. Immature and mature schizonts were found in hepatocytes and the parasite was associated with generalized hepatic necrosis. By TEM, schizonts divided by endopolygeny, and merozoites lacked rhoptries. Molecular characterization of parasites present in liver and brain tissues at the cox1 gene showed a high degree of identity (97-98%) and clustered together with Sarcocystis canis, S. lutrae, S. arctica, S. speeri, S. turdusi, and S. rileyi in a phylogenetic study. This is the first report of S. canis-like infection from Asia.

Phase transition of plasma membranes of rat hepatocyte and hepatoma cells by electron diffraction.

The transition temperatures (Tc) of the plasma membranes of Reuber H-35 hepatoma and normal ACl rat hepatocytes were measured by electron diffraction under physiological conditions. Diffraction rings below Tc indicate the existence of solid lipid domains. The Tc of both membranes increase to a plateau value of 17.5 degrees during the storage period of 4 days at 4 degrees, the rate of increase being slower for normal liver membranes. The extrapolations to zero storage suggest an innate difference of Tc for these two membranes. Storage in oxygen-reduced media slows down the initial increase in Tc in normal liver membranes, while the depletion of divalent cations accelerates the increase. Differences in lipid composition are partly responsible for this behavior.

Protection of the membrane calcium adenosine triphosphatase by cholesterol from thermal inactivation.

There is correlative evidence that one mechanism of cellular thermoresistance is an increased level of membrane cholesterol. The hypothesis that cholesterol protects membrane proteins from thermal inactivation was tested using Ca-ATPase as a model. The intracellular Ca2+- and Mg2+-dependent ATPase from muscle sarcoplasmic reticulum was reconstituted into lipid mixtures containing different amounts of cholesterol [cholesterol/phospholipid molar ratio (C/PL) = 0.1 or 0.3]. The rate of thermal inactivation of calcium uptake activity of the reconstituted vesicles with C/PL = 0.3 was found to be significantly lower than those with C/PL = 0.1 in the temperature range 43-47 degrees C where hyperthermic cell killing occurs. At 43 degrees C, this is equivalent to a 3 degrees C temperature shift. ATP hydrolysis of Ca-ATPase was found to be substantially heat resistant in reconstituted vesicles with C/PL = 0.1 or 0.3. Glycerol (10%) protects while ethanol (2.5%) and the local anesthetics dibucaine, tetracaine, and procaine sensitize the thermal inactivation of calcium uptake. To investigate the molecular mechanisms of thermal inactivation and cholesterol protection, the responses of the physical state of the lipid and protein conformation to hyperthermic sensitizers and protector were monitored using fluorescent and spin label probes and circular dichroism, respectively. The calcium uptake inactivation appears to be due to a direct thermotropic conformational change (denaturation) of the protein. Cholesterol raises the temperature of inactivation, as does glycerol, while ethanol and the local anesthetics lower it.

Formulation, stability, and antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugate of thioether phospholipid.

1-beta-D-Arabinofuranosylcytosine 5'-diphosphate-rac-1-S-octadecyl-2-O- palmitoyl-1-thioglycerol (ara-CDP-DL-PTBA) is an effective stable 1-beta-D-arabinofuranosylcytosine (ara-C) conjugate of thioether phospholipid against a variety of transplantable tumors in mice. The conjugate was formulated in a micellar solution by sonication, in which the conjugate exists as micellar discs (size, 0.01 to 0.04 micron). Analyses on thin-layer and high-pressure liquid chromatography showed that the conjugate was chemically stable upon storage at 3-4 degrees C for more than a 6-mo period. However, stored at room temperature for 3 mo it began to degrade (3 to 11%) to 1-beta-D-arabinofuranosylcytosine 5'-monophosphate and phosphatidic acid. At 3-4 degrees C, the micellar structure remained generally unchanged for 6 mo (size, less than 0.1 micron). Samples stored for 4 mo at room temperature formed some larger vesicles (size, 0.1 to 0.4 micron). Antitumor activity against i.p. implanted L1210 leukemia in mice remained relatively constant with samples stored for 6 mo at 3-4 degrees C or 3 mo at room temperature. 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (ara-CTP) levels were elevated (greater than 500 pmol/10(7) cells) in L1210 leukemia cells within 1 h following i.p. administration of 400 mg/kg of ara-CDP-DL-PTBA to mice. More importantly, retention of cellular ara-CTP was prolonged (greater than 24 h) in these tumor cells as compared with ara-C treatments. Administration of ara-CDP-DL-PTBA to mice with colon 26 carcinoma (s.c.) resulted in both significant antitumor activity with an increased life span greater than 100% and decreased tumor size. The conjugate also demonstrated a dose-dependent therapeutic effect in mice with M5076 sarcoma (s.c.) as demonstrated by decreases in tumor size and liver metastases. Overall, ara-CDP-DL-PTBA, a stable lipid conjugate of ara-C in a micellar solution, appears to offer substantial therapeutic benefit to mice with leukemia and solid tumors warranting its further development and clinical investigation.

Dielectrophoresis of cell-size liposomes.

The dielectrophoresis (DEP) behavior of cell size liposomes were studied in the frequency range from 20 kHz to 3 MHz. Liposomes in the size of about 10 microns in diameter were made from egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), egg phosphatidylglycerol (PG) and brain phosphatidylserine (PS). These liposomes, having an internal conductivity of 58 microS/cm, were suspended in either PEG or Ficoll solutions of conductivities 9 and 20 microS/cm, respectively. The liposomes were induced to form pearl chains in an electric field gradient in specially designed chambers with either coaxial or flat electrodes. Liposome rotation and convection (mixed positive and negative dielectrophoresis) were observed just beyond the experimental frequency range, within which positive dielectrophoresis leading to pearl chain formation were found. The threshold voltages that caused pearl chain formation were recorded within the experimental frequency range. The threshold voltages remained more or less constant within the positive DEP frequency range, and increased at both ends of the range. Charged liposomes (PS, PG) were found to have lower threshold than uncharged ones (PC, PE). Theoretical DEP spectra were calculated using a model proposed by Kaler and Jones ((1990) Biophys. J. 57, 173-182), for uncharged liposomes. The surface current effect in charged liposomes was accounted for, using an approximation proposed by Schwartz ((1962) J. Phys. Chem. 66, 2636-42). The experimental data agreed with theoretical prediction in general. The higher cutoff frequencies observed experimentally were thought to represent a slight lowering of internal conductivity due to leakage. The higher than predicted ratio between threshold voltages of charged and uncharged liposomes was interpreted to be due to the slight difference in size of these two types of liposomes. The agreement between theory and experiment showed that the available theory was adequate, and that liposomes provide a good model to study the nature of dielectrophoretic forces.

Chemical co-treatments and intramembrane particle patching in the poly(ethylene glycol)-induced fusion of turkey and human erythrocytes.

Several chemical co-treatments were used to lower the threshold concentrations of poly(ethylene glycol) (PEG) required to induce fusion between turkey erythrocytes and between human erythrocytes. Concanavalin A was used in conjunction with 25% (w/w) PEG to induce turkey erythrocyte fusion. The fusion percentage increased with increasing concentrations of concanavalin A and the duration of concanavalin A treatment. In samples with high percentages of fusion, numerous hemispherical intramembrane particle-free zones (bubbles) in the plasma membrane were revealed by freeze-fracture electron microscopy. However, concanavalin A treatment did not facilitate fusion between human erythrocytes even at 35% PEG, although slight intramembrane particle patching was observed under this condition. Spermidine (0.05% w/v), trichloroacetic acid (100 mM) and ethanol (4% v/v) were found to promote fusion of human erythrocytes in 25% PEG. In all of these cases, intramembrane particle patching was observed by freeze-fracture electron microscopy in the presence of PEG. When applied alone, only ethanol caused a slight intramembrane particle patching. Neither dimethylsulfoxide (2% v/v), lysophosphatidylcholine (lysoPC, 0.15 mM), nor polylysine (mol. wt. 1000-4000, 0.05% w/v) promoted fusion of human erythrocyte in 25% PEG. None of these chemical treatments, alone, or in combination with PEG, caused intramembrane particle patching. We conclude that the positive effect of chemical treatments on PEG-induced cell fusion is closely related to the formation of intramembrane particle-free zones on the plasma membrane.

Merocyanine interaction with phosphatidylcholine bilayers.

Merocyanine (MC 540) is a fluorescent probe whose optical properties depend on the environmental polarity. In the presence of lipid bilayers, MC 540 binds to the membrane surface while simultaneously changing its fluorescence properties. Previous studies have shown that the fluorescence of merocyanine depends upon the lipid packing in the membrane. We measured the partitioning of MC 540 and its fluorescence properties in the presence of phosphatidylcholine membranes. We found that the fluorescence of MC 540 shows, as expected, a major change around the main phase transition of phosphatidylcholine membranes. However, instead of a step-like increase of fluorescence, the maximum at phase transition was observed. We were able to explain our data by combining two effects; dependence of MC 540 fluorescence on temperature and lipid fluidity. In addition, we established that the increase of the fluorescence intensity in the presence of lipid bilayers in the fluid state is due to the elevated partitioning of the probe into the lipid phase. The partition of MC 540 into the fluid membrane does not depends on the dye concentration in the aqueous phase. When lipid was in the gel phase the partitioning of the dye increased with its bulk concentration, whereas the fluorescence intensity remained unchanged. We conclude, therefore, that MC 540 forms nonfluorescent complexes when in the gel lipid membrane.

Merocyanine 540 as a probe to monitor the molecular packing of phosphatidylcholine: a monolayer epifluorescence microscopy and spectroscopy study.

The characteristics of the fluorescent dye, merocyanine 540 (MC-540), incorporated in monolayers of 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied in different states of molecular packing. Conditions for phase separation in these monolayers were defined by their pressure/area (pi-A) isotherms. Within the liquid expanded (LE) and the liquid condensed (LC) coexisting phases of DPPC monolayers, low light level epifluorescence microscopy revealed 'dark' discoid domains embedded in a 'bright' matrix. Under the same conditions, and at temperatures as low as 12 degrees C, the pi-A isotherms of POPC demonstrate the existence of a single phase, and no fluorescent domains were observed. Fluorescence spectra of MC-540 labelled monolayers, recorded in different structural states, reveal three distinct emission peaks: a 572 nm peak, present for monolayer packing conditions at low surface pressures; a 585 nm peak, similar to that obtained from dye molecules in fluid phase lipid bilayers, and observed here within the respective area/molecule ranges of 54-62 A2 and 62-69 A2 for monolayers of DPPC and POPC with diminishing intensity at increasing surface pressure; and finally, a peak at 560 nm, which predominates in densely packed POPC monolayers. Our results are interpreted on the basis of dye partitioning between monolayer and subphase, and different orientations of the dye with respect to the monolayer in various structural states. The usefulness of MC-540 to differentiate lipid packing in cell membranes is discussed.

Poly(ethylene glycol)-conjugated surfactants promote or inhibit aggregation of phospholipids.

The calcium-induced aggregation of dilauroyl phosphatidic acid (DLPA) suspensions, with or without added poly(ethylene oxide) (PEO)-conjugated surfactants containing 4 to 30 ethylene oxide subunits, were monitored by turbidity measurement and quasi-elastic light scattering (QLS). The aggregation was inhibited (protected) by the incorporated PEO surfactant for most samples, while a window for promotive effect was found for samples with low surface coverage by the PEO moiety of the incorporated surfactant. Promotion occurs only when the aggregation is slow and at a low level. The promotion is explained by the synergistic effect of PEO and divalent calcium cations when the steric repulsion is weak. The promotion/protection crossover is a display between the PEO/calcium synergistic effect and the steric repulsion.

The effect of lipid molecular packing stress on cationic liposome-induced rabbit erythrocyte fusion.

The effect of curvature stress on the efficiency of cationic liposome-induced fusion between rabbit erythrocytes was studied. Multilamellar cationic liposomes containing 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and different PEs (1,2-dilnoleoyl-sn-glycero-3-phosphoethanolamine (dilin-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and lysophosphatidylethanolamine, egg (lyso-PE)) were used to induce cell-cell fusion. It was found that high cell-cell fusion yield (FY) of about 50% could be achieved in sucrose solution by using cationic liposomes containing 50% DOTAP. Cell-cell fusion was assayed by shape criterion and was verified by fluorescence microscopy, using a membrane dye mixing method. The curvature stress, as a result of mixing unsaturated PEs in cationic liposomes, had a significant effect on cell-cell FY which increased with the degree of acyl chain unsaturation, in the order dilin-PE > DOPE > POPE > lyso-PE. Replacement of dilin-PE, DOPE, or POPE by lyso-PE gradually in cationic liposomes lowered the cell-cell FY from 50% to 1%. Furthermore, cationic liposome induced cell lysis, and fusion between cationic liposomes and cells, as assayed by the N-(lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rh-PE/NBD-PE) energy transfer method, followed the same order as that for cell-cell fusion. Fusion between the negatively charged PS liposomes and cationic liposomes also followed the same order. The electric double layer screening by electrolytes in NaCl-containing solution and phosphate buffered saline (PBS) was found to reduce the cell-cell FY and cell lysis. These findings suggest that liposome-induced cell-cell fusion was achieved by cationic liposomes serving as fusion-bridges among cells.

Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures. Structural changes between hexagonal, cubic and bilayer phases.

The polymorphic phase behaviour of dilinoleoylphosphatidyethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is investigated by freeze-fracture electron microscopy, X-ray diffraction and 31P-NMR. The structures at 5% or less POPC are predominantly inverted hexagonal (HII), whereas at 15% or more POPC, the structure is mostly bilayer (L), interrupted by defects (lipidic particles). A cubic phase structure is observed in the transition range between H and L phases; the cubic arrangement deteriorates at higher temperatures into an amorphous aggregate of spherical units. Both cubic and amorphous structures contribute to the isotropic 31P resonance, with no preference for PC or PE partitioning in the isotropic motion as observed by high resolution NMR. The existence of the cubic phase seems to depend cirtically on the homogeneity and the degree unsaturation of the phospholipids.

Electron diffraction studies of human erythrocyte membrane and its lipid extracts. Effects of hydration, temperature and hydrolysis.

The organization of lipid molecules in individual human erythrocyte ghost membranes and single bilayers of their total lipid extracts were studied by low-dose electron diffraction in a controlled environment. The highest onset temperature (Ts) at which diffraction rings corresponding to a gel state appeared, were found to be in the range of -2 to -4 degrees C for both the whole ghost membrane and bilayers of its total lipid extracts. The onsets were abolished by dehydration before separated crystallizations of cholesterol and phospholipid occurred. Ts increased as a result of free fatty acids accumulation in membranes after phospholipase A2 treatment or storage.

Selective decoration of hydrophilic moieties of membrane molecules in freeze fracture.

Selective decoration of the hydrophilic moieties of phospholipid molecules on freeze fractured bilayer faces was made using residue water vapor in an oil-free vacuum unit. The preferential decoration technique was applied to label structural faults of bilayers, such as domain boundaries and other regions of molecular dislocation which are not visible by conventional morphological observations.

Electrofusion of cell-size liposomes.

Cell size liposomes of egg phosphatidylcholine (PC), trans-acylated egg phosphatidylethanolamine (PE), bovine brain phosphatidylserine (PS) and egg phosphatidylglycerol, suspended in 2.5% of polyethylene glycol (M(r) 8000), Ficoll (M(r) 400,000) or Dextran (M(r) 71,200) were aligned by dielectrophoresis and fused by applying a 1.7 kV/cm pulse of varying duration. Because the internal and external media of liposome suspension can be controlled, and the surface charge is known, the results can be described mathematically. The fusion yields (FY) at different pulse length were measured by microscopy. The FY curves were sigmoidal, with a common minimally required pulse length of 19 microseconds, and shifted to longer pulse length with increasing external media conductivity or vesicle surface charge. The types of polymer in solutions had little effect. The minimal required pulse length was interpreted to be the minimum rise time for the smallest vesicle capable of reaching the bilayer breakdown potential induced by the pulse, and the sigmoidal shape FY curves represented the cumulative vesicle size distribution curve. The shifting of FY curves upon changing media conductivity or vesicle surface charge were quantitatively accounted for by the balance of pulse-induced dipole-dipole attraction and electrostatic repulsion. Deviation from sigmoidal shape FY curves in the cases of charged liposomes was explained by increasing electrostatic repulsion due to vesicle deformation under pulse-induced dipole pressure. The data confirm the hypothesis that membrane potential breakdown is a pre-requisite or minimum requirement for electrofusion, and support our earlier proposition that pulse-induced dipole force plays an important role in the electrofusion process, and that electrostatic repulsion posts additional barrier to electrofusion.