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A new method for the preparation of liposomes is described that avoids the use of pharmaceutically unacceptable solvents and energy-expensive procedures such as sonication. The method is based on the initial formation of a proliposome mixture containing lipid, ethanol and water, which is converted to lipsomes by a simple dilution step. Measurements using 6-carboxyfluorescein as a marker indicate that water-soluble drugs can be trapped with extremely high efficiency (65-80% depending on lipid composition). The structural organization of the proliposome mixture and the final liposomes were characterized using electron microscopy and 31P-NMR.

We showed that insulin appears in the chick embryo before beta-cells are recognizable, as well as in the egg constituents even before fertilization. Acid-ethanol extracts of 2- to 8-day-old embryos were gel-filtered on Sephadex G-50. The peak of immunoreactive insulin chromatographed in a position corresponding to that of authentic insulin. The immunoreactive insulin extracted from embryos was approximately 2 ng/g wet wt during early embryogenesis (days 2, 3, and 4), with a 2- to 3- fold increase by days 5 and 6, in conjunction with pancreatic development. The heads of the embryos contributed 22-23% of the total insulin on days 3 and 4, but only 5% by day 5. Based on its reactivity in a pork insulin RIA, chicken insulin RIA, and rat adipocyte bioassay, we concluded that the material is very similar to avian (chicken or turkey) rather than mammalian type insulin. Similar immunological and biological insulin-like activity [but at much lower concentrations (0.2-0.8 ng/ml)] were recovered from the gel-filtered acid-ethanol extracts of yolk and white of unfertilized and fertilized eggs. This study, which shows that insulin is present at a very early stage in ontogeny, extends observations that insulin is native to organisms that lack pancreatic islets, including flies, worms, and microbes.

The association constants of hen egg yolk riboflavin binding protein with 8-substituted riboflavins were measured using a fluorometric titration method in the range of 10-40 degrees C, and thermodynamic parameters were calculated using ordinary methods. The flavins used were riboflavins whose 8-methyl group was substituted with HRN, RR'N, RO type groups, or halogen atoms. From a plot of delta H degrees against delta Su (unitary entropy change), the flavins were classified into four groups, i.e., HRN, RR'N, RO, and halogen type substituents. In each group, the linear free energy relationship, delta H degrees = u . delta Su+v (u and v were constants specific for each group), was found to be similar to that for egg white riboflavin binding protein, though with different specific constants for each egg protein. The relation between delta Su and the bulkiness (molecular volume) of 8-substituents suggested burying of the 8-substituents in a cavity of the protein similar to that of the egg white protein, but of smaller volume. The behavior of the halogen group was similar to that of the other three groups, and could be explained in terms of the bulkiness of the substituents rather than by assuming electric repulsion between halogen substituents and the binding site, which is in contrast with the case of egg white protein. A linear relation between v value and the wavelength of the visible absorption peak of flavins wa also found.

A hyperlipidemic control serum can be simple prepared from animal lipid sources. Beta- and pre-beta-lipoproteins containing cholesterol and triglyceride are removed from porcine serum by treatment with dextran sulfate and calcium ions. A triglyceride-rich fraction containing only trace amounts of cholesterol is isolated from chicken egg-yolks. The two fractions are then combined in 40 mmol/L sodium bicarbonate to give the desired values for cholesterol and triglyceride. The preparation is stabilized against surface denaturation during long-term storage at 5 degrees C perhaps for as long as two years, by adding 0.25 g of Triton X-100 surfactant per liter, and against an accidental exposure to short-term freezing by adding 10 g of sucrose per liter. We used this solution as a diluent to reconstitute lyophilized bovine serum. The resulting product, having been prepared from only animal sources, is free of hepatitis-associated constituents, and is remarkably clear, homogeneous, and stable. Results obtained with it are precise.

A new sensitive assay method for sphingoids, 4-D-hydroxysphinganine, sphingosine, and sphinganine, involving reversed phase HPLC was described. Chromatography of p-N-nitrophenylacetyl derivatives of the sphingoids showed sufficient resolution, and each peak showed a linear relationship between molar quantity and area response, from 10 pmol to 10 nmol. The method was applied to the analysis of long chain bases in intestinal glycolipids from embryonic and adult Japanese quails. At the embryonic stage of 12 days' incubation, 4-D-hydroxysphinganine accounted for about 40% of the long chain bases of glycolipids, a concentration comparable to that in adult tissue. Egg yolk, which is an exclusive exogenous nutrient mixture supplied by the mother, contained only a few percent of 4-D-hydroxysphinganine as a glycolipid component. While, [3H]palmitic acid administered to embryos was incorporated into 4-D-hydroxysphinganine as well as sphingosine and sphinganine. These results suggest that sphingoids of intestinal glycolipids including 4-D-hydroxysphinganine are synthesized in the tissue, excluding the possibility of their exogenous origin in the embryonic tissue.

The amino acid sequence of hen egg yolk-riboflavin binding protein (yolk-RBP) was determined by conventional methods. The sequence was identical with that of hen egg white-riboflavin binding protein except that their carboxyltermini were different, that of yolk-RBP lacked 11 or 13 amino acid residues, while hen plasma-RBP had the same C-terminal sequence as white-RBP. This indicated that the C-terminal 11 or 13 amino acid residues in plasma-RBP might be cleaved off during the incorporation from the blood into the oocyte or in the yolk fluid. Yolk-RBP had the same characteristics as white-RBP, such as N-terminal pyroglutamic acid, polymorphism in the amino acid sequence (Lys/Asn) at the fourteenth residue from the N-terminal end, carbohydrate chains attached to both Asn(36) and Asn(147) residues, and phosphate groups bound to some serine residues in the sequence of Ser(185) to Ser(197) as a cluster. These results led us to the conclusion that yolk- and white-RBPs are bio-synthesized from the same gene in the different organs (liver and oviduct). The carbohydrate composition of yolk-RBP was identical to that of plasma-RBP but different from that of white-RBP showing that the processing of the carbohydrate chains in the liver was different from that in the oviduct.