After prolonged exposure to ascorbate, collagen synthesis in cultured human skin fibroblasts increased approximately 8-fold with no significant change in synthesis ofnoncollagen protein. This effect of ascorbate appears to be unrelated to its cofactor function in collagen hydroxylation. The collagenous protein secreted in the absence of added ascorbate was normal in hydroxylysine but was mildly deficient in hydroxyproline. In parallel experiments, lysine hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) activity increased 3-fold in response to ascorbate administration whereas proline hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) activity decreased considerably. These results suggest that collagen polypeptide synthesis, posttranslational hydroxylations, and activities of the two bydroxylases are independently regulated by ascorbate.Ascorbic acid is essential for normal collagen formation (1-3) by virtue ofthe fact that it is a required component in the synthesis of hydroxyproline and hydroxylysine in collagen (4). Hydroxyproline serves to stabilize the collagen triple helix (5, 6); its absence results in structurally unstable collagen (7, 8) which is not secreted from cells at a normal rate (9). Hydroxylysine is necessary for formation of the intermolecular crosslinks in collagen (10). In addition, specific carbohydrate residues are linked glycosidically to collagen through hydroxylysine, a process that may be important in the regulation of crosslink formation (11).It is generally believed that ascorbate modulates collagen production through its effect on prolyl hydroxylation (12). There have been indications, however, that ascorbate may have an additional role in collagen biosynthesis (13-16). Notable are the early studies by Jeffrey and Martin (13) who observed a substantial increase in the size of chicken long bones cultured in the presence of ascorbate, concomitant with an increase in the incorporation of proline into peptidyl hydroxyproline.In this study we have examined the long-term effect of ascorbate on collagen production by cultured human skin fibroblasts. The influence ofascorbate on prolyl hydroxylase (prolylglycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2 oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) levels was also examined simultaneously to understand better the interrelationship of collagen synthesis and posttranslational hydroxylation. The data indicate that ascorbate increases collagen synthesis by acting at a level other than hydroxylation. MATERIALS AND METHODSHuman skin fibroblasts from a normal 3-day-old boy (GM 970) were obtained from the Institute for Medical Research (Camden, NJ) and grown to confluent density in Dulbecco's modified Eagle's medium buffered with 24 mM sodium bicarbonate and 25 mM Hepes and supplemented with 20% fetal calf serum (GIBCO) which had been inactivated for 30 min at 560C. Cultures were growth arrested for 90 hr in ...

The conversion of testosterone to 5 alpha-dihydrotestosterone by prostate particulates from rats, dogs, and humans was investigated, and significant species differences were found with their pH profiles, affinities for 4-azasteroidal inhibitors, and sensitivities to mercuric sulfhydryl reagents. The pH optima for the rat (pH 7), the dog (pH 6), and the human (pH 5) enzyme are significantly different. Mersalyl acid and p-hydroxymercuribenzoate inactivate only the rat 5 alpha-reductase, but not the human or dog enzyme. The rank orders of potencies of 24 3-oxo-4-azasteroids to inhibit 5 alpha-reductases of the rat, dog, and human prostate are different. The variation of the 17 beta-functional groups of the inhibitors demonstrates clearly the species differences. Those inhibitors with a 17 beta-diethylcarbamoyl, 17 beta-diisopropylcarbamoyl, 17 beta-t-butylcarbamoyl, or 17 beta-secbutylcarbonyl functional group are approximately equipotent as inhibitors of the rat and human enzymes, whereas they are only 0.1-15% as potent as inhibitors of the dog enzyme. On the other hand, those inhibitors with a 17 beta-spiroether functional group are most potent as inhibitors of the rat enzyme, are 15-50% as potent as inhibitors of the dog enzyme, and are 0.2-0.4% as potent as inhibitors of the human enzyme. Those inhibitors with a 17 beta-n-octylcarbamoyl, 17 beta-(1-carboxyethyl), or 17 beta-(1-carboxy-3-butyl) functional group are 2-3 orders of magnitude less potent as inhibitors of the dog and human enzymes than as inhibitors of the rat enzyme. These results suggest that prostatic 5 alpha-reductases of rats, dogs, and humans are significantly different. In spite of the significant species differences in inhibitor affinities, where determined, inhibition of the rat, dog and human enzymes by these compounds is competitive with testosterone. These 3-oxo-4-azasteroids have a similar rank order of potency as inhibitors of 5 alpha-reductase in human normal, benign hyperplastic, and cancerous prostates, indicating that the inhibitor-binding sites of 5 alpha-reductase in the prostate in different pathological states are similar. The affinities of the 3-oxo-4-azasteroids for rat prostatic cytosol receptor were determined. Five of these 5 alpha-reductase inhibitors have no significant affinity for the androgen receptor, whereas others do have an affinity for the receptor.

A series of steroids, primarily 4-azasteroids, were prepared and tested in vitro as inhibitors of human and rat prostatic 5 alpha-reductase and of binding of dihydrotestosterone to the rat androgen receptor. The primary structural modifications were changes of the A ring and of moieties attached at the C-17 position of the steroid nucleus. New A-ring modifications included the 4-cyano-3-oxo-delta 4 system in the carbocyclic series and 1 alpha-CN, 1 alpha-CH3, 1 alpha,2 alpha-CH2, 2 beta-F, 2-aza, 2-oxa, and A-homo changes in the 3-oxo-4-aza series. In addition, 4-azasteroids with a D-homo ring or methyl substitution at C-7 (alpha and beta) or C-16 (alpha and beta) were prepared. The majority of the C-17 substituents were prepared from reactive intermediates derived from the 17 beta-COOH. Enhanced 5 alpha-reductase inhibition in both the human and rat enzyme assays is seen with 4-CN substitution on 3-oxo-delta 4 steroids and with a C-17 side chain incorporating a lipophilically substituted semipolar group on the 4-aza-3-oxo-5 alpha-androstane nucleus. Fewer highly active compounds were found in the human enzyme assay than in the rat assay. Structural requirements for inhibition of the rat androgen receptor are much different from those for inhibition of the enzyme. The 17 beta-OH moiety enhances potency more than any other feature while introduction of double bonds at C-1 or C-5 in the azasteroid gives a small improvement. Azasteroids unsubstituted at the 4-position show greatly diminished receptor activity.

Mixtures of 1 -methyl-3-ethylimidazolium chloride and aluminum chloride form salts that are liquid at room temperature. As the proportions of organic chloride and aluminum chloride are varied, the 13C NMR chemical shifts of the cation carbons change and the 27A1 line widths of the chloroaluminate anions change. The 13C shifts may be explained by anion-cation interactions and the27A1 line widths by the presence of more than one type of chloroaluminate anion. Temperature-dependent 27A1 NMR results show chemical exchange between chloroaluminate anions in the aluminum chloride rich molten salts.

A series of A-ring heterocyclic steroids has been prepared and tested for inhibition of rat prostatic steroid 5 alpha-reductase in vitro. Strikingly high inhibitory activity was found with a group of 17 beta-substituted 4-methyl-4-aza-5 alpha-androstan-3-ones. These compounds were prepared from 3-keto-delta 4-precursors by oxidative (O3 or NaIO4-KMnO4) A-ring cleavage followed, in turn, by ring closure with an amine and hydrogenation over platinum catalyst. Other A-ring azasteroids were made by Beckmann rearrangement of oximes of 2-oxo-A-nor, 3-oxo- and 4-oxo-5 alpha-androstanes. An A-nor-2-oxo-3-azasteroid was prepared by oxidative decarbonylation of a precursor 2,3-dioxo-4-azasteroid with m-chloroperbenzoic acid. A-ring modifications of the 4-azasteroids included delta 1-unsaturation, 2- and 4-substituents, and 3-carbonyl replacements. Side chains at the 17-position were varied with an emphasis on carboxylate derivatives (salts, esters, and amides).