Biomedical subjects

S W Hui

Publications and source records attributed to S W Hui.

At least 73 records · Page 4Linked to original sources

PubMed1991

Bilayer packing stress and defects in mixed dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine and their susceptibility to phospholipase A2.

The hydrolysis of mixed dilinoleoylphosphatidylethanolamine (DiLinPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) dispersions by porcine phospholipase A2, under conditions leading to the bilayer-to-nonbilayer phase transition, has been studied. Two structurally distinct forms of the dispersions were used, multilamellar vesicles (MLV) and supercritical large unilamellar vesicles (SCLUV). In MLV, maximum free fatty acid was produced in dispersions containing 85 mol % DiLinPE. The peak in the fatty acid release is found at the onset of appearance of the nonbilayer defects reported earlier. DiLinPE was found to be preferentially hydrolyzed as compared to POPC. When cholesterol was added to the mixed DiLinPE/POPC MLV, the onset of the observable appearance of nonbilayer defects, the positions of the peaks for total hydrolysis, and the preferential hydrolysis of DiLinPE were all shifted toward lower DiLinPE concentrations. In SCLUV, where the appearance of nonbilayer structures is prevented by constraining the lipids in bilayer configuration, the hydrolysis by PLA2 increases with increasing DiLinPE as predicted from the increase in the calculated monolayer bending energy. The results are interpreted to be related to the pretransition molecular-packing stress and defects at the onset of the bilayer-to-nonbilayer transition. Results indicate that the porcine pancreatic phospholipase A2 activity is controlled by bilayer-packing stress, which may cause structural defects of the substrate, among other factors. Results also indicate a preferential localization of PE at stress-related defect regions.

Animals↗

PubMed1991

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.

Lipid Bilayers↗

PubMed1991

Effects of retinyl acetate on surface morphology and intramembrane particle distribution in the plasma membrane of 10T1/2 cells.

Scanning electron microscopy and freeze fracture electron microscopy were used to characterize membrane ultrastructural differences between parental, C3H/10T1/2, and carcinogen-initiated, INIT C3H/10T1/2, cells and treatments with retinyl acetate. The intramembranous particle distribution on the E-face was detected and quantitated by the methods of automated image analysis to obtain statistically meaningful numerical characteristics of intramembranous particle size and density. Subtle differences were found when no differences were apparent by light microscopy or by scanning electron microscopy. Initial retinyl acetate treatment caused a significant increase of the intramembranous particle size in parental cells. Intramembranous particle density increased for retinyl acetate treatment in parental and INIT cells and in INIT cells previously maintained but withheld from retinyl acetate. Intramembranous particle distribution analysis includes the interparticle distance of nearest neighbors and the randomness of the distribution by the differential density distribution function, which compares the observed sample to Poisson modified for particle size. These measures show that the three cell groups that have been treated with retinyl acetate have a more even distribution of intramembranous particles than was found for untreated parental cells. The relationship between the freeze fracture morphology and the biological responses to retinyl acetate treatment is discussed.

Animals↗

PubMed1991

Dipole interactions in electrofusion. Contributions of membrane potential and effective dipole interaction pressures.

The contributions of pulse-induced dipole-dipole interaction to the total pressure acting normal to the membranes of closely positioned pronase treated human erythrocytes during electrofusion was calculated. The total pressure was modeled as the sum of pressures arising from membrane potential and dipole-dipole attraction opposed by interbilayer repulsion. The dipole-dipole interaction was derived from the experimentally obtained cell polarizability. The threshold electric field amplitude necessary for fusion of pronase-treated human erythrocytes was experimentally obtained at various combinations of pulse duration, frequency, and the conductivity of the external medium. The theoretical values of the critical electric field amplitude compared favorably to the experimentally obtained threshold field amplitudes. Fusion by dc pulses may be primarily attributed to attainment of sufficiently high membrane potentials. However, with decreasing external conductivity and increasing sinusoidal pulse frequency (100 kHz-2.5 MHz), the induced dipole-dipole interactions provide the principal driving force for membrane failure leading to fusion.

Electric Stimulation↗

PubMed1991

The mitogenic activities of phosphatidate are acyl-chain-length dependent and calcium independent in C3H/10T1/2 cells.

Phosphatidates (PA or phosphatidic acid) were shown to have mitogenic properties, including the stimulation of DNA synthesis and calcium mobilization in C3H/10T1/2 cells. Their continuous presence for a minimum of 7 h induced DNA synthesis with kinetics similar to that observed when 10% fetal bovine serum was used as a mitogen. PAs with long chain saturated fatty acid moieties were more mitogenic, in a dose-dependent fashion, than PAs with short saturated or unsaturated fatty acid moieties. When compared with lysostearoyl-PA (LSPA), distearoyl-PA (DSPA) was as potent with respect to the induction of DNA synthesis. Lysooleoyl-PA (LOPA) was slightly more potent than dioleoyl-PA (DOPA), but much weaker than DSPA and LSPA. Preincubation with dilauroyl-PA (DLPA) reduces the mitogenic effect of DSPA by 85%. The pattern of mitogenic inhibition suggests that a chain-length-independent, yet PA-specific, mechanism is involved. Both DSPA and DLPA are equally taken up by the cells after 30 min. LOPA, but not LSPA, produced a large calcium transient (1.3 microM), which we found to be derived from intracellular sources. DSPA, the most mitogenic PA tested, produced a weaker transient (0.6 microM). Interestingly, LSPA did not produce any detectable calcium transient. These results suggest that the chain-length-specific step in the signaling mechanism of PA occurs after the initial chain-length-independent partitioning and/or binding to the membrane and that the induction of DNA synthesis is not related to the observed calcium transients.

Animals↗

PubMed1990

Effect of unsaturated phosphatidylethanolamine on the chain order profile of bilayers at the onset of the hexagonal phase transition. A 2H NMR study.

The quadrupolar splitting profiles of methylene groups along the acyl chains of perdeuteriated dimyristoylphosphatidylcholine (DMPC-d54) in mixtures with dioleoylphosphatidylethanolamine (DOPE) were studied by 2H NMR. The quadrupolar splittings, obtained for lipid mixtures in the bilayer state, were measured as functions of temperature and PE:PC ratio and were used to obtain the approximate gauche probabilities at a given chain position, pB. Ratios (R) of pB for C13, C12, and C11 relative to that of the plateau region were used to characterize the effect of increasing PE on the gauche content of PC chains. At all temperatures studied (including the bilayer to hexagonal phase transition region), for each ratio R (e.g., RC13/P), the relative gauche content of the DMPC chains was similar over the range of 25-85% PE. DOPE is viewed in simple terms as having a "conical" shape; if this geometry applies to the acyl chain region of the molecule, a greater lateral pressure would be expected toward the center of the bilayer as the PE content is increased, resulting in a decreased gauche content, relative to the plateau, of those methylene groups of PC. The failure to observe the predicted increase in lateral pressure has ramifications for the cone-shape molecular model. The overall "cone shape" of PE is seen to arise from the smaller size of the head-group relative to the acyl chains; however, the acyl chain region itself is not rigidly cone-shaped and is better represented by a flexible "balloon". These results were supported by small-angle X-ray diffraction, which showed a decreasing trend in the area per molecule with increasing PE content.

Deuterium↗

PubMed1990

Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures.

We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.

Calcium↗

PubMed1990

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols.

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.

Diglycerides↗

PubMed1990

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.

Animals↗

PubMed1990

Electron microscopic observation of the aggregation of membrane proteins in human erythrocyte by melittin.

Human erythrocytes and erythrocyte ghost membranes were treated with native and modified melittins, up to 250 nmol/mg membrane protein. Native melittin induced aggregation of intramembranous particles (IMPs, observed by freeze-fracture electron microscopy), and created large, smooth bilayer areas devoid of IMP. The degree of IMP aggregation increased with increasing concentration of melittin, corresponding to hemolysis results. Membrane ghosts were slightly more susceptible to IMP aggregation than membranes on intact cells. The potency of inducing IMP aggregation was ranked in the order of: native melittin greater than acetylated melittin greater than succinylated melittin = 0. The concentration range of melittin which caused IMP aggregation corresponded to that which caused the immobilization of band 3 proteins as detected by measurement of rotational mobility by transient dichroism (Dufton et al. (1984) Eur. J. Biophys. 11, 17-24). Because both IMP aggregation and band 3 protein immobilization decreased with decreasing positive charge of the melittins used, the nature of melittin-protein interaction is likely to be at least in part electrostatic in the case of human erythrocyte membranes. Possible roles of IMP aggregation and the consequent creation of 'exposed' bilayer areas in the cytotoxic reaction of melittins are discussed.

Bee Venoms↗

PubMed1990

Fluorescence measurements of fusion between human erythrocytes induced by poly(ethylene glycol).

The kinetics of poly(ethylene glycol) (PEG)-induced fusion between intact human erythrocytes was continuously monitored by a fluorescence lipid mixing method, utilizing the dequenching of the fluorescence probe, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl ] phosphatidylcholine (C12-NBD-PC). The steady-state fluorescence intensity was detected from the surface of cells in a monolayer on an alcian blue-coated glass coverslip. The relief of fluorescence self-quenching after fusion between C12-NBD-PC labeled and unlabeled intact erythrocytes was measured. The extent of fluorescence dequenching was normalized based on the measured concentration of probes in membranes, the projected partial dequenching due both to dilution by intercellular fusion, and the dilution between the inner and outer leaflets of membranes (flip-flop). There was no significant increase in fluorescence intensity during PEG treatment of 5 min, at 4 degrees C. Intensity increased immediately after the dilution of PEG, and reached saturation in 30 min. The efficiency of fusion increased with the increasing of PEG concentrations. Only 4% enhancement of saturated relative fluorescence intensity was detected in 25 wt% PEG-induced cell fusion; 23% enhancement in 30 wt%; and 66% enhancement in 35 wt%. The transfer of fluorescent probes between membrane bilayer leaflets (flip-flop) was also monitored during the fusion process. Flip-flop was monitored in confluent monolayers as well as in isolated cells. There was no significant spontaneous flip-flop within 30 min of dilution. The relative fluorescence intensity enhancement contributed by the dilution of probes between fused labeled and unlabeled cells (at a 1:1 ratio) was found to account for only 39% of the observed final dequenching, whereas the contribution by flip-flop associated with cell fusion was found to account for 9%, and flip-flop without fusion contributed approximately 18%. A portion of the flip-flop is a consequence of hemolysis. Therefore, fluorescence dequenching measurements of fusion of whole cells must be interpreted with caution.

Biophysical Phenomena↗

PubMed1990

Effect of lateral mobility of fluorescent probes in lipid mixing assays of cell fusion.

Monolayers of human erythrocytes, immobilized on a cover slip, were induced to fuse by polyethylene glycol (mol wt 8,000). The mobility of fluorescent probes, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadizol-4-yl)amino]dodecanoyl] phosphatidyl-choline (C12-NBD-PC), from labeled cells to unlabeled cells was monitored by video-enhanced fluorescence microscopy. A dequenching curve was obtained from the measurement of fluorescence intensities of pairs of fused cells over time. The dequenching curve and the curve obtained from macroscopic measurements of a cell monolayer (described in the preceding article) were compared and discussed. The slow probe transfer rate between a pair of fused cells was explained by a diffusion model based on membrane area conservation and the geometry of the fusion lumen. An equivalent lumen between two fused cells, thought to be the main rate limitation of probe mobility after fusion, was calculated to be approximately 130 nm in diameter. Lumens of 75 nm in diameter were observed by electron microscopy. Thus, the rate of macroscopic fluorescence dequenching depends not only upon the fusion efficiency, but also upon the number of simultaneous fusion partners, the geometry of their contact points, and the lateral mobility of the fluorescent probes through these points. The relative fusion efficiency can be derived only from the saturation dequenching values.

4-Chloro-7-nitrobenzofurazan↗

PubMed1990

Effects of pulse length and pulse strength on transfection by electroporation.

The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.

DNA↗

PubMed1989

Induction of c-fos and c-myc proto-oncogene expression by epidermal growth factor and transforming growth factor alpha is calcium-independent.

Intracellular calcium has been proposed to be an important mediator of signal transduction by various growth factors. We have studied the role of intracellular calcium in the mitogenic stimulation of C3H 10T1/2 mouse fibroblasts by epidermal growth factor and transforming growth factor alpha. We have found that both these peptides can cause a marked, transient increase in intracellular calcium levels. This rise occurs only in the presence of extracellular calcium. However, this calcium transient is not involved in the accumulation of c-fos and c-myc mRNAs which are elicited by these growth factors, since mRNA induction is observed to an equivalent degree in the absence or presence of extracellular calcium. These results demonstrate that although these growth factors cause an increase in intracellular calcium, the calcium second messenger system is not responsible for the induction of c-fos and c-myc mRNAs in C3H 10T1/2 fibroblasts.

Animals↗

PubMed1989

Electron diffraction and diffraction contrast imaging of thin organic films.

The applications of electron diffraction and diffraction contrast electron microscopy with which to study the structure and dynamics of organic thin films are discussed. The techniques of making thin film deposits on substrates and of forming free-standing thin films over holes on the substrate are described. Selected area electron diffraction and diffraction contrast imaging techniques for thin film studies are elaborated, and examples are given. Methods to reduce radiation damage and environmental protection of the thin film specimen are outlined. The interpretation of electron diffraction and imaging data is given for three cases: 1) The effects of film tilting and molecular tilting (with respect to the film plane) are examined. 2) The detection of phase transition is illustrated. 3) The use of labels to mark film domains is shown together with the measurement of dynamic movement.

Chemistry, Organic↗

PubMed1989

Effects of lipid packing on polymorphic phase behavior and membrane properties.

The self assembly of phospholipid molecules in the bilayer form was considered in terms of equivalent molecular shapes representing intermolecular forces. The equivalent size of each phospholipid headgroup was approximated by the net atomic volume plus the volume of the associated water molecules, which was derived from water/hydrocarbon partitioning experiments. The equivalent lengths of unsaturated acyl chains were derived from the retention time data from chromatographic measurements. The spontaneous curvature of various phospholipid monolayers was calculated from their equivalent molecular shapes, and the energy required to flatten them to the bilayer plane was calculated, using the known bending modulus. With increasing bending energy, the mixtures showed increasing susceptibility to phospholipase A2, facilitated lipid transfer rate by phospholipid exchange proteins, permeability to carboxyfluorescein, incorporation of human erythrocyte proteins, and calcium transport by Ca-ATPase from sarcoplasmic reticulum in reconstituted vesicles. When the calculation was applied to known lipid compositions of nine cellular membranes, the protein/lipid ratio and phospholipid/cholesterol ratio were found to have a positive and a negative correlation, respectively, with the latent bending energy of the phospholipids. The energy expense in conforming to a bilayer phase may be an important physical parameter regarding the activity and the biogenesis of membranes.

Cholesterol↗

PubMed1988

Electron microscopic study of the calcium phosphate-induced aggregation and membrane destabilization of cytoskeleton-free erythrocyte vesicles.

Cytoskeleton-free vesicles derived from human erythrocytes were treated with trypsin, chymotrypsin, or neuraminidase followed by calcium, phosphate, or combined calcium/phosphate treatments in order to study the roles of cell surface proteins and glycoproteins in calcium/phosphate-induced cell aggregation and fusion. Vesicle aggregation (a necessary pre-cursor to membrane fusion) and subsequent membrane destabilization (an essential component of fusion) were examined by freeze-fracture electron microscopy. Enzymatic treatment alone had no effect on the morphology of the cytoskeleton-free vesicles. Neither did separate calcium nor phosphate treatments, although the treatment of the cytoskeleton-free vesicles with calcium did reduce their size slightly. Enzymatic pretreatment had no effect on the calcium-induced size changes. In contrast, the combination of calcium and phosphate drastically disrupted the membrane integrity of aggregated cytoskeleton-free vesicles at pH 7.8, although the effect was reduced at lower pH values. The extent of this membrane destabilization was independent of enzyme treatment. Our results indicate: (1) that the cell surface proteins and glycoproteins have only secondary effects on calcium/phosphate-induced cell aggregation and membrane destabilization, (2) that these processes primarily depend on the reaction between calcium and phosphate ions at the membrane surface, and (3) that cytoskeletal elements probably play no active (positive) role in the Ca2+/PO4(3-) induced erythrocyte membrane fusion process, apart from maintaining cell shape.

Calcium↗

PubMed1988

Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry.

Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners.

Calcium↗