Light scattering and turbidity measurements on lipid vesicles

The dynamic behaviour of model membranes in the form of sonicated liposomes in excess water was studied by means of 90 °C light scattering and turbidity measurements. Computer calculations based on the Rayleigh-Gans theory of light scattering were used to estimate the average size of lipid vesicles dispersed in water, taking into account the various structures of the vesicles. Normal reversible changes in the scattered light intensity and turbidity with temperature could be accounted for mainly by the change in the refractive index of the lipid and irreversible anomalous changes were explained on the basis of fusion of smaller aggregated vesicles.     

Interaction of Nucleic Acids with Lipid Membranes

Abstract

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg- lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5° to 74°C, the transition is broadened from 0.7°C to 2.7°C. Varying the phosphatidyl-serine-lecithin-ratio from 0–100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5°C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed. At present, the biological relevance of the results may be more in the drastic decrease in cooperativity than in the slight modulation of the stability. From nearly 180 base pairs opening up cooperatively in free nucleic acid this number is lowered to less than 50, a size in the range of promotor regions.     

Temperature dependence of optical properties of aqueous dispersions of phosphatidylcholine

Aqueous dispersions of dipalmitoyl phosphatidyl choline exhibit a sharp decrease in turbidity at the crystal-liquid phase transition temperature of 41°C. The intensity of light scattered at 45°, 90°, and 135° also undergoes a sharp drop at the same temperature. Similarly, the refractive index of such dispersions decreases abruptly with the phase transition. Employing the relationship between light scattering intensity and specific refractive increment, it can be shown that about one half of the change in absorbancy and scattering are accounted for by the change in refractive index. The change in refractive index can be entirely accounted for by the known expansion and corresponding decrease in density of the bilayer. That part of the observed change in scattering and turbidity which is not accounted for by the observed change in average refractive index is apparently due to a decrease in the anisotropy of the bilayer during the melting process. Calculations based on a model which, although oversimplified, is consistent with the known thinning of the bilayer during the melting process, give quantitative agreement with experimental results. Below the phase transition temperature other changes in optical properties are observed; near 32°C, the light scattering envelope changes and the turbidity of dispersions drops markedly. The average refractive index remains constant in this region. For this and other reasons, it is postulated that these pre-transition changes indicate an aggregation-disaggregation phenomenon.     

Light scattering study of phosphatidylcholine vesicles at the pretransition

Laser light scattering has been used to investigate the thermal pretransition of dipalmitoylglycerophosphocholine vesicles with variable radius as obtained by the mild sonication method. Intensity changes in 90° scattered light are observed at the pretransition for larger vesicles and actually increase with increasing vesicle size, reaching a constant value.This constant value is in good agreement with the value calculated from the refractive index data.The intensity ratio of scattered light at temperatures of 30°C and 40°C (I₄₀/I₃₀) approaches unity at a radius of small single-bilayer vesicle. This result is interpreted as no pretransition for small vesicles in agreement with the calorimetric results. An expression of the particle scattering factor is also presented for multilayered shells composed of anisotropic elements. It is shown numerically, using this expression, that changes in the lipid layer thickness and the tilting angles at the pretransition have no effects on the scattering factor. Therefore it is concluded that the intensity changes in scattered light reflect the changes in the refractive index of the vesicle originating in the polar head groups.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Kinetics on the turbidity change of wheat starch during its retrogradation

Wheat starch dispersions of 10–40% (w/w) were gelatinized and the change in turbidity of each solution during storage was measured in the 400–1100 nm wavelength range. The relative transmittance, defined as the ratio of transmittance at any storage time to that at the initial time, decreased when the solutions were stored at 5 and 30 °C; the decrease, reflecting the progress of retrogradation, was larger at 5 °C than at 30 °C. Most of the changes in relative transmission taking place over 14 days were achieved during the first 90 min. The change in the relative transmittance is inversely proportional to the energy required for deformation. The kinetics on change in relative transmittance can be expressed by Weibull equation. The larger rate constant at higher starch concentration could be ascribed to the state of the starch granules, which depended on starch concentration.     

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

DISINTEGRATION OF THE CELLS OF SIPHONOUS GREEN ALGA CODIUM EDULE (BRYOPSIDALES,CHLOROPHYTA) UNDER MILD HEAT STRESS1

The siphonous green alga Codium edule P. C. Silva (Bryopsidales, Chlorophyta) has the highest covering ratio among the macroalgae on the coral reef of Nanwan Bay in southern Taiwan, but its population in the subtidal region drastically decreases from July to September each year. The objective of this study was to determine whether the high temperature of summer could be the basis for this population decrease. Chlorophyll fluorescence measurements revealed that when the algae were incubated at 35°C (a temperature that can be reached in southern Taiwan during the summer), their photosynthetic activities were almost completely inhibited after about 8 h. The circadian rhythm of photosynthesis was disrupted at a temperature as low as 32°C. TEM studies showed that 4 h incubation at 35°C induced a decrease in turgidity accompanied by vacuole shrinkage and plasmolysis. The marked disintegrative changes, including damage to organelles, such as chloroplasts and nuclei, occurred after about 8 h, at which time central vacuoles collapsed and the cell interior was then filled with numerous small vesicles. Our results suggested that the rise in seawater temperature during the summer could be one of the major causes of the massive death of C. edule in the field.     

Effect of temperature on the ultrastructure of bovine oocytes in culture

Cattle oocytes were cultured at 35, 37 and 39°C to determine the effect of temperature on the maturation and degeneration of the cells, as indicated by changes in the ultrastructure of the cytoplasm. The culture temperature influences the characteristics of oocyte peripheral cytoplasm. A temperature of 35°C was most favourable, as indicated by the highest frequency of granulated vesicles. Growth rate of oocytes, measured by width of the perivitelline space and the distribution of cortical granules, is a little slower at 35° C than at 37 or 39° C. However, myelin bodies are less numerous at 35°C, which seems to indicate that this temperature is more favourable than higher temperatures. Higher frequency of myelin bodies could indicate a more advanced stage of metabolism or quicker degenerative changes in the cells.     

Heat Treatment and β-Carotene Incorporation Effect in the Interaction of β-Lactoglobulin and Carboxymethylcellulose System

Encapsulation technologies using proteins or polysaccharides can be employed with the purpose of solubilizing and protecting carotenoids. However, information on the role of protein and polysaccharide interactions is still slightly limited. The aim of this work was to investigate the effect of β-carotene linked to protein β-lactoglobulin (BLG) in the interaction carboxymethylcellulose (CMC) using isothermal titration calorimetry (ITC). Firstly, BLG and CMC interaction was assessed by means of turbidity analysis. Based on the results of turbidity, the thermodynamic profile of BLG-CMC complexes at pH 4.0 was obtained using ITC analysis at 25 °C. Afterward, it was evaluated the effect of a thermal treatment applied to the BLG (68 °C for 50 min) in the interaction with CMC also using ITC and circular dichroism (CD). ITC and CD analysis showed that the heat treatment applied on BLG did not cause changes in molecular interactions. The binding isotherm of BLG-CMC complexes incorporated with β-carotene showed an increase in the molar ratio and a slight decrease in enthalpy of the system. Incorporation of β-carotene in the system did not significantly affect the BLG and CMC interaction, suggesting this system can be applied in food application as encapsulation.     

Kinetic and thermodynamic studies of the fusion of small unilamellar phospholipid vesicles

Small phospholipid vesicles, prepared so as to minimize impurities, fuse relatively slowly resulting in the time-dependent development of a characteristic endotherm in differential scanning calorimetry and corresponding changes in the Raman spectrum. The stability of small vesicles towards fusion increases with increasing acyl chain length for the series C-14 through 18. Within the protocols of these experiments, the fusion rate remains unchanged whether the vesicles are held at 10°C below T_m or at T_m itself. We have determined enthalpies of transition for small vesicles and fusion product for C-14 through C-18. In each case ΔH for small vesicles is lower than that of the corresponding multilamellar vesicles, while the fusion product ΔH is intermediate between small and multilamellar vesicles. The apparent lack of concensus in the literature as to the nature of the fusion process is ascribed to the variety of protocols used as well as the presence or absence of fusion-inducing impurities.     

Temperature-Induced Changes in the Number of Vesicles in the Free Nerve Endings of Temperature Neurons of the Snake

By observing ultrastructural changes under the electron microscope, we illustrated exocytosis and recycling of vesicles in the infrared receptor, a kind of free nerve ending in the pit organ of the crotaline snake, Trimeresurus flavoviridis. While maintaining the snake pit organs at stable temperatures of 15°C, 25°C, and 30°C, we fixed them by perfusion and then processed them for transmission electron microscopy. The largest number of clear and coated vesicles appeared in the terminals at the lowest temperature. The perimeter and area of a terminal were enlarged at 30°C, and “opening waves” on the plasma were prominently found at the highest temperature. We also observed coated vesicles that budded from the plasma membrane in the terminals. The configuration of mitochondria in the terminals was quantitatively different between lower and higher temperatures. The data suggest that exocytosis and endocytosis in these terminals operate in a manner similar to that observed in other cell types.     

Evaluation of a New Extrusion Device for the Production of Stable Oligolamellar Liposomes in a Liter Scale

Abstract

A procedure for large-scale production of extruded oligolamellar liposomes was developed. the extrusion technique described is rapid, simple, and reproducible. Vesicles with a diameter in the range of 200-500 nm are obtained. the liposomal preparations were characterized by their specific turbidity (determined spectrophotometrically) and contamination with large particles (> 1 µm). Liposome physical stability was determined using a parameter (P) that relates the ratio of the specific turbidity before and after standardized centrifugation.

The extruded oligolamellar liposomes obtained were stable, and after 4 weeks storage at 4°C no irreversible aggregation or fusion of the vesicles occurs.

The extrusion process ensures sterilization of the final liposomal product, making it acceptable as a pharmaceutical preparation for parenteral use.     

Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry

The interaction of the surfactant octyl glucoside (OG) with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and soy bean phosphatidylcholine (soy bean PC) was studied using high-sensitivity titration calorimetry. We determined the partition coefficient of OG between water and lipid bilayers and the transfer enthalpy of the surfactant by addition of lipid vesicles to OG monomers or vice versa. Comparison with the micellization enthalpy of the surfactant gives information on differences in the hydrophobic environment of OG in a liquid-crystalline bilayer or a micelle. The average partition coefficient P in mole fraction units for x_e≈0.12–0.2 decreases slightly from 4152 at 27°C to 3479 at 70°C for DMPC and from 4260 to 3879 for soy bean PC, respectively. The transfer enthalpy ΔH^T of OG into lipid vesicles is positive at 27°C and negative at 70°C. Its temperature dependence is larger for the incorporation of OG into DMPC than into soy bean PC vesicles. It is concluded that OG in DMPC vesicles is better shielded from water than in soy bean PC vesicles or in micelles. Titration calorimetry was also used to determine the phase boundaries of the coexistence region of mixed vesicles and mixed micelles in the systems OG/DMPC, OG/DPPC, OG/DSPC, and OG/soy bean PC vesicles at 70°C in the liquid-crystalline phase. DMPC and soy bean PC solubilization was also studied at 27°C to investigate the effect of temperature. The effective surfactant to lipid ratios at saturation, R_e^sat, for all PCs studied are in the range between 1.33–1.72 and the ratios at complete solubilization, R_e^sol, are between 1.79–3.06. At 70°C, the R_e^sat values decrease with increasing chain length of the saturated PC. The ratios depend also slightly on temperature and the degree of unsaturation of the fatty acyl chains. For the OG/soy bean PC system, the coexistence range for mixed vesicles and mixed micelles is larger than for the corresponding PCs with saturated chains.     

Pressure-induced Shape Change of Phospholipid Vesicles: Implication of Compression and Phase Transition

A microscopic study has allowed the analysis of modifications of various shapes acquired by phospholipid vesicles during a hydrostatic pressure treatment of up to 300 MPa. Giant vesicles of dimyristoylphosphatidylcholine / phosphatidylserine (DMPC/PS) prepared at 40°C mainly presented a shape change resembling budding during pressure release. This comportment was reinforced by the incorporation of 1,2-dioleyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or by higher temperature (60°C) processing. The thermotropic main phase transition (Lα to Pβ′) of the different vesicles prepared was determined under pressure through a spectrofluorimetric study of 6-dodecanoyl-2-dimethylamino-naphtalene (Laurdan) incorporated into the vesicles’ bilayer. This analysis was performed by microfluorescence observation of single vesicles. The phase transition was found to begin at about 80 MPa and 120 MPa for DMPC/PS vesicles at, respectively, 40°C and 60°C. At 60°C the liquid-to-gel transition phase was not complete within 250 MPa. Addition of DMPE at 40°C does not significantly shift the onset boundary of the phase transition but extends the transition region. At 40°C, the gel phase was obtained at, respectively, 110 MPa and 160 MPa for DMPC/PS and DMPC/PS/DOPE vesicles. In comparing volume data obtained from image analysis and Laurdan signal, we assume the shape change is a consequence of the difference between lateral compressibility of the membrane and bulk water. The phase transition contributes to the membrane compression but seems not necessary to induce shape change of vesicles. The high compressibility of the Lα phase at 60°C allows induction on DMPC/PS vesicles of a morphological transition without phase change.     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Temperature-dependent transmembrane potential changes in cells infected with a temperature-sensitive moloney sarcoma virus

Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33°C and a normal morphology at 39°C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C₃-(5). Fluorescence was inversely correlated with Em. Cells at 33°C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33°C to 39°C in both directions, increasing in the 33°C to 39°C direction and decreasing in the 39°C to 33°C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33°C to 39°C direction. Shifting from 39°C to 33°C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33°C to 39°C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.     

NMR study of rapid water diffusion across lipid bilayers in dipalmitoyl lecithin vesicles

A method for measurement of rapid diffusional exchange between external and internal water in lecithin vesicles is described. Paramagnetic ions were inserted inside DPL vesicles and the NMR relaxation times for water protons were measured as a function of temperature. It was found that water diffusion rate is described by a single activation energy of 15±1 kcal/mole in the temperature range 16 – 35°C and exhibits a maximum at 44°C. The permeability of DPL vesicles to water was calculated to 16–18 × 10^?4 cm/s at 44°C and 1.7 × 10^?4 cm/s at 20°C.     

Membranolysis by the ninth component of human complement

The lytic property of isolated C9 was shown by using heat (48°C) to induce polymerization of C9, which was then exposed to egg lecithin vesicles containing carboxyfluorescein. In this environment, C9 penetrated the vesicles and released the marker. C5b-9 and C5b-8 also released carboxyfluorescein at 48°C, as did C5b-7 to a lesser extent. However, neither isolated C5b-6, C7, C8 nor albumin caused such release. At 30°C, the C9 remained in the monomeric form and could not lyse the vesicles.C9 polymers formed at 48°C were ring-shaped with an internal diameter of approximately 100 Å and an outer diameter of approximately 180 Å. These rings of C9 polymers strikingly resembled the ultrastructures formed by the membrane attack complex of complement, viewed from the top.Our results indicate that polymeric C9 causes the membranolysis of phospholipid vesicles and, hence, that C9 alone is a cytotoxic molecule.     

Phase transitions in phospholipid vesicles Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol

We have studied the solid to liquid-crystalline phase transition of sonicated vesicles of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine. The transition was studied by both fluorescence polarization of perylene embedded in the vesicles, and by the efflux rate of trapped ²²Na⁺.Fluorescence polarization generally decreases with temperature, showing an inflection in the region 32–42°C with a mid-point of approximately 37.5 °C. On the other hand, the perylene fluorescence intensity increases abruptly in this region. To explain this result, we have proposed that, for $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ where $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ is the transition temperature, perylene is excluded from the hydrocarbon interior of the membranes, whereas, $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ this probe may be accommodated in the membrane interior to a large extent.The self-diffusion rates of ²²Na⁺ through dipalmitoylphosphatidylglycerol vesicles exhibit a complex dependence on temperature. There is an initial large increase in diffusion rates (approximately 100-fold) between 30 and 38 °C, followed by a decrease (approximately 4-fold) between 38 and 48 °C. A monotonic increase is then observed at temperatures higher than 48 °C. The local maximum of ²²Na⁺ self-diffusion rates at approximately 38 °C coincides with the mid-point of phase transition as detected by changes in fluorescence polarization of perylene with the same vesicles. Vesicles composed of dipalmitoylphosphatidylcholine show the same general behavior in terms of ²²Na⁺ self-diffusion rates at different temperatures, except that the local maximum occurs at approximately 42 °C.The temperature dependence of the permeability and the appearance of a local maximum at the phase transition region could be explained in terms of a domain structure within the plane of the membranes. This explanation is based on the possibility that boundary regions between liquid and solid domains would exhibit relatively high permeability to ²²Na⁺.Mixed vesicles composed of equimolar amounts of dipalmitoyl phospholipids and cholesterol show no abrupt changes in the temperature dependence of either perylene fluorescence polarization or ²²Na⁺ diffusion rate measurements. This is taken to indicate the absence of agross phase transition in the presence of cholesterol.