John T. Ho

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol)

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the rigidization of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.

Apparatus for High-Resolution Birefringence Measurement in Liquid Crystals

Abstract A rotating-analyzer technique for measuring the optical birefringence in liquid crystals is described. This apparatus is continuously readable and has excellent sensitivity. For a sample 50 μm thick, the apparatus has a resolution in the change of birefringence of 4 × 10−6, corresponding to a temperature change of 1 mK in the sample tested.

Dynamic light scattering study of calcium-induced fusion in phospholipid vesicles

Acidic sonicated phospholipid vesicles can undergo dramatic morphological changes due to fusion in the presence of divalent metal ions. For example, small spherical phosphatidylserine vesicles can form scroll-like cylinders which precipitate in the presence of Ca2+ above a threshold concentration. Subsequent addition of EDTA will yield large, unilamellar vesicles. These events have previously been established through the combined use of differential scanning calorimetry and freeze-fracture electron microscopy. We have applied the technique of dynamic light scattering to follow these fusion events rapidly, accurately, and non-perturbatively as they occur in solution at calcium concentrations slightly below threshold for precipitation.

Detection of δ-aminolevulinic acid, porphobilinogen and porphyrins related to heme biosynthesis by high-performance liquid chromatography

Abstract Two new high-performance liquid chromatographic methods are described for the quantitative determination of porphyrins and their precursors. In our method, sub-nanomole quantities of prophyrins, δ-aminolevulinic acid and porphobilinogen derivatized with o -phthalaldehyde were injected onto a C 18 reversed-phase column and eluted with 0.1 M monobasic sodium phosphate—methanol—tetrahydrofuran (4:6:3) and detected with a spectrofluorometer. A second reversed-phase system using methanol—tetrahydrofuran— 22 m M acetate buffer (15:6:11) was also developed.

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca2+-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca2+ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca2+ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca2+ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca2+ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca2+, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca2+ concentration, and identical to that with pure phosphatidylserine in excess Ca2+. The results imply that Ca2+- induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.

Quantitative determination of porphyrins, their precursors and zinc protoporphyrin in whole blood and dried blood by high-performance liquid chromatography with fluorimetric detection

Methods for the determination of porphyrins, delta-aminolevulinic acid (ALA), porphobilinogen (PBG) and zinc protoporphyrin of heme biosynthesis in whole blood and dried blood are described. Erythrocyte porphyrins and the precursors ALA and PBG were extracted from whole blood (50 microliter) with 0.3 ml of methanol and 1.5 M hydrochloric acid (2:1, v/v). Zinc protoporphyrin was extracted with an acetone-pyridine-Sterox solution. Other major interfering metabolites were removed by centrifugation. An aliquot of the supernatant was injected onto the reversed-phase C18 column for detection of porphyrins with excitation wavelength at 405 nm and emission wavelength at 630 nm. The mobile phase was 0.1 M phosphate-methanol-tetrahydrofuran (18:30:16, v/v/v), pH 5.38. The ALA and PBG were derivatized with o-phthalaldehyde before injection. The detection excitation wavelength occurred at 330 nm and the emission wavelength at 418 nm. The mobile phase was 0.1 M phosphate-methanol (7.5:5), pH 3.38. For the dried blood specimen of filter paper, two 0.64-cm discs punched out from the blood-impregnated filter paper were placed in a test tube containing 200 microliter of 0.9% saline for 60 min or longer at room temperature and then treated as whole blood.

Raman study of calcium-induced fusion and molecular segregation of phosphatidylserine/dimyristoyl phosphatidylcholine-d54 membranes

Raman spectroscopy has been used to study the effect of Ca2+ on the molecular properties of model membranes consisting of mistures of phosphatidylserine and dimyristoyl phosphatidylcholine-d54. The I2880/I2935 intensity ratio associated with the C-H stretching modes is used to monitor the phosphatidylserine molecules, while the linewidth at 2103 cm-1 associated with the C-2H stretching modes is used for the dimyristoyl phosphatidyl-choline-d54 molecules. Membranes containing phosphatidylserine and dimyristoyl phosphatidylcholine-d54 at a molar ratio of 1 : 2 show evidence of initial immiscibility, which is further enhanced by the addition of Ca2+. Membranes containing phosphatidylserine and dimyristoyl phosphatidylcholine-d54 at a molar ratio of 2 : 1 are completely miscible, and fuse with the addition of Ca2+. The phosphatidylserine molecules in the fused product of mixed vesicles have highly rigid acyl chains, and behave identically to those in an earlier Raman study of Ca2+-induced fusion of pure phosphatidylserine vesicles. The dimyristoyl phosphatidylcholine-d54 molecules are also solid-like, suggesting a higher degree of miscibility with phosphatidylserine in the presence of Ca2+ than dipalmitoyl or distearoyl phosphatidylcholine.

The anomaly of sedimentation at normal gravity in microemulsions

Etudes sur lexemple de microemulsions constituees de docusate de Na (variete purifiee dAerosol OT), dethanol, doctane, deau et de NaCl

Cholesteric Pitch Near the Smectic-C Phase

Abstract The temperature dependence of the cholesteric pitch of the chiral ester 4-n-hexyloxyphenyl-4-(2”-methylbutyl)biphenyl-4-carboxylate has been measured in detail using the method of selective reflection. This compound has a cholesteric to smectic-C transition at 79°C. Because of the first-order nature of the transition, the pretransitional unwinding of the pitch is considerably less pronounced than that in cholesteryl esters near the smectic-A transition. The wavelength of maximum reflection increases from 440 nm at 110°C to 560 nm at the transition. When fitted to a power-law temperature dependence, the data imply an exponent of 0.82 ± 0.15, which is consistent with de Gennes’ theory in the meanfield approximation.

Cholesteric pitch of cholesteryl decanoate near the smectic-a transition

Abstract The temperature dependence of the cholesteric pitch in cholesteryl decanoate has been measured near the smectic-A transition. The data show a power-law divergence with a critical exponent v = 0.67 ± 0.03, in agreement with previous results in cholesteryl nonanoate.