Incorporation of excess cholesterol by high density serum lipoproteins.

Excess cholesterol was added to human HDL3 and to bovine mammalian high density serum lipoprotein (HDL) by incubating aqueous lipoprotein solutions with solid dispersions of [4-(14)C]cholesterol on Celite. Lipoprotein cholesterol complexes were isolated by centrifugation and filtration through a Sepharose 4B column. The pure complexes were analyzed for protein and lipid content and composition and were subsequently investigated by physical methods (analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy), in order to detect any structural changes induced by added cholesterol. The rates of cholesterol uptake varied as an inverse function of the intrinsic cholesterol present in the native lipoproteins. The maximum cholesterol taken up by human HDL3 increased the free cholesterol content from 3--4% (initial) up to 22% of the total lipoprotein weight. Bovine HDL was observed to increase its free cholesterol content from 2--4% (initial) up to 11--17% of the total lipoprotein weight, before denaturation. At maximum levels of added cholesterol, both lipoproteins had increased molecular weights and sedimentation velocity coefficients corresponding to the increased mass of the particles. No major changes in the hydrodynamic properties were observed. At the molecular level, the protein components only showed a 15--20% decrease in fluorescence intensity, possibly a consequence of a modified environment of the aromatic amino acid residues. In the human HDL3, added cholesterol increased the microviscosity of the lipid domains by 1.2 P at 25 degrees C (from 3.4 to 4.6 P), but did not affect the fluidity of bovine HDL lipids (5.9 P).     

Gonadal Steroid Regulation of Growth-Associated Protein GAP-43 mRNA Expression in Axotomized Hamster Facial Motor Neurons

Treatment with testosterone propionate (TP) after nerve injury is known to accelerate both the rate of axonal regeneration and functional recovery from facial paralysis in the adult male hamster. Peripheral nerve injury is also known to increase the expression of a 43 kilodalton growth-associated protein (GAP-43). In the intact brain, GAP-43 expression is affected by gonadal steroids. We thus postulated that steroidal modulation of GAP-43 gene expression may be a component of the neurotrophic action of TP in regenerating neurons. This issue was examined in hamster facial motor neurons (FMN) which contain androgen receptors and which have been shown to respond to exogenous steroids in a number of previous studies. Castrated adult male hamsters were subjected to right facial nerve transection and treated with either TP via subcutaneous hormone capsule implants, or left untreated (no hormone replacement). At post-injury/treatment times of 0.25, 2, 4, 7, and 14 d, the brain stem regions were harvested, cryostat sections were collected through the facial motor nucleus, and in situ hybridization was done using a ³³P-labeled GAP-43 cDNA probe. Quantitative analysis of the autoradiograms by computer assisted grain counting revealed that axotomy produced a dramatic increase in GAP-43 mRNA levels in FMN by 2 d post-axotomy and that this increase remained through 14 d post-injury in both the TP-treated and the untreated group. In the nonhormone-treated group, there was a statistically significant dip in GAP-43 mRNA levels in FMN at 7 d post-operative, relative to 4 d post-operative levels. TP-treatment prevented this transient decline in GAP-43 mRNA levels in axotomized FMN.     

Androgen receptor mRNA regulation in adult male and female hamster facial motoneurons: effects of axotomy and exogenous androgens

Testosterone propionate (TP) administration at the time of facial nerve injury in the adult hamster augments the regenerative properties of the injured facial motoneurons (FMN), with the androgen receptor (AR) playing a key role in mediating the actions of TP on facial nerve regeneration. The purpose of the present study was to determine the effects of axotomy on AR mRNA expression in FMN. This was accomplished using in situ hybridization in conjunction with a (35)S-labeled AR riboprobe. Gonadally intact adult male and gonadectomized (gdx) adult female hamsters were subjected to a right facial nerve axotomy, with the left side serving as internal, unoperated control. Half the animals were subcutaneously implanted with a 10-mm TP Silastic capsule, and the other half were sham-implanted. An additional group of nonaxotomized, gonadally intact males was also included. Postaxotomy survival times were 1, 4, and 7 days. At 1 postoperative day 1, there were no effects of axotomy on AR mRNA levels. By postoperative days 4 and 7, axotomy caused a significant decrease in AR mRNA levels in FMN of gonadally intact males, relative to either the contralateral control FMN of the same animals or FMN from the group of gonadally intact males that were not subjected to facial nerve axotomy. There were no significant differences between AR mRNA levels in contralateral control FMN and FMN from the gonadally intact group of nonaxotomized males. TP administration at the time of axotomy had no effect on AR mRNA levels in either the axotomized or contrala(teral control FMN of gonadally intact males, relative to the nonaxotomized, gonadally intact male group. Corroborating our previous work, AR mRNA levels were reduced in the contralateral control FMN of gdx females, relative to the nonaxotomized, gonadally intact male group, with axotomy having no additional effects. The data are discussed in a mechanistic framework suggesting how TP acts to augment facial nerve regeneration.     

Fluorescence polarization studies of human and rhesus A-I apolipoproteins and their complexes with phosphatidylcholine.

Human and rhesus A-I apolipoproteins, covalently labelled with dansyl chloride, were used in fluorescence polarization studies of: 1) the monomeric structure of the free proteins in solution; 2) the interaction of the apolipoproteins with sonicated egg phosphatidylcholine; and 3) the size of the saturated complexes of protein with phospholipid. The results indicate that both monomeric apolipoproteins have relatively rigid, yet asymmetrical structures, with Stokes radii of 24.2 ± 0.5 Å, in neutral aqueous solutions. Axial ratios are of the order of $\text{6}\text{1}$ or $\text{4}\text{1}$ for hydrated, prolate or oblate ellipsoids, respectively. A molar excess of about 200 phosphatidylcholine molecules are required to saturate each apolipoprotein. At saturation, the complexes with both proteins have Stokes radii of 40.6 ± 1.7 Å. Since the radius of phosphatidylcholine vesicles is around 125 Å, we conclude that the complexes are relatively small structures derived from disruption of the lipid vesicles, rather than from adsorption of the proteins on intact vesicles.