Enzyme replacement via liposomes. Variations in lipid compositions determine liposomal integrity in biological fluids.

Liposomes survive exposure to biological fluids poorly, extruding trapped enzymes, drugs, or solutes upon interaction with serum or plasma constituents. We have quantified the disruptive effects of human serum on liposomes and have studied whether various modifications in their phospholipid composition might produce liposomes with an increased carrier potential for application in vivo. Multilamellar liposomes (phosphatidycholine 70:dicetyl phosphate 20:cholesterol 10) were prepared with 3H-labeled phosphatidylcholine as the lipid phase marker and [14C]inulin and horseradish peroxidase as aqueous phase markers. Gel exclusion chromatography showed that 32 +/- 3% of [14C]inulin and 27 +/- 7% of horseradish peroxidase were lost after 1 h incubation with 10% (v/v) human serum. Loss of aqueous solutes was reduced to 20 +/- 5%/h and 17 +/- 2%/h, respectively, after treatment with decomplemented serum (56 degrees C, 30 min). Loss induced by serum was concentration and time dependent: to 57 +/- 2% at 1 h and 67 +/- 14% at 24 h, with 50% serum; plasma was slightly less perturbing whereas human serum albumin was not at all disruptive. By incorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of [14c]-inulin in the presence of 10% serum was reduced to 12 +/- 4%/h; increasing the molar percentage of cholesterol to 35% also stabilized the lipid bilayers, reducing leakage to 20 +/- 7%/h. Both small and large unilamellar vesicles could not be stablilized against serum-mediated leakage by the incorporation of sphingomyelin. The data suggest that cholesterol and sphingomyelin enhance liposomal integrity in the presence of serum or plasma and promise to yield enhanced survival of drug-laden lipid vesicles in biological fluids in vivo.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

Physical reasons for secondary structure stability: alpha-helices in short peptides

It was recently found that some short peptides (including C- and S-peptide fragments of RNase A) can have considerable helicity in solution, ^1–12 which was considered to be surprising. Does the observed helicity require a new explanation, or is it consistent with previous understanding? In this work we show that this helicity is consistent with the physical theory of secondary structure^12–19 based on an extension of the conventional Zimm-Bragg model.²⁰ Without any special modifications, this theory explains reasonably well almost all the experimentally observed dependencies of helicity on pH, temperature, and amino acid replacements. We conclude that the observed “general level” of helicity of C- and S-peptides (5–30% at room temperature and 10–50% near 0°C) is “normal” for short peptides consisting mainly of helix-forming and helix-indifferent residues. The helicity is modified by a multitude of weak specific side chain interactions, many of which are taken into account by the present theory;^13–19 some discrepancies between the theory and experiment can be explained by weak side-chain-side chain interactions that were neglected. A reasonable coincidence of the theory with experiment suggests that it had been used to investigate the role of local interactions in the formation of α-helical “embryos” in unfolded protein chains.     

DNA-binding proteins related to the dosage of specific yeast chromosomes

A comparison has been made of the $\text{in vitro}$ DNA-binding proteins of specific aneuploid and isogenic euploid cells of $\text{Saccharomyces cerevisiae}$ by DNA-cellulose chromatography. We have been able to detect changes in the level of a small fraction of the yeast DNA-binding proteins which can be related to the dosage of specific yeast chromosomes. At least four proteins show a dosage related to the cellular level of chromosome I and at least one protein shows a dosage related to the level of chromosome VI.