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The synthesis and aromatase inhibitory profile of 6 alpha and 6 beta-propargyl androstenedione and estrenedione are described. The targeted compounds 1 and 2 were prepared by addition of the propargyl Gringard to the 5 alpha,6 alpha-epoxy bisketal 6 or the 5 alpha,6 alpha-epoxy diacetate 7 followed by dehydration of the 6 beta-propargyl 5 alpha-hydroxy diones 10 and 11 using thionyl chloride. Treatment of the 6 beta-propargyl analogs 1 and 2 with hydrochloric acid gave the corresponding 6 alpha-propargyl isomers 3 and 4. Inhibitory activity of the synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H]-4-androstenedione as substrate. Under initial velocity assay condition of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent KiS ranging from 10 to 66 nM, with the Km for androstenedione being 55 nM. 6 alpha-Propargylandrost-4-ene-3,17-dione and 6 alpha-propargylestr-4-ene-3,17-dione were found to be potent competitive inhibitors of aromatase (Ki 37 and 66 nM, respectively). On the other hand the 6 beta-propargylandrost-4-ene-3,17-dione (6 beta-PAD) and 6 beta-propargylestr-4-ene-3,17-dione (6 beta-PED) were found to bind to aromatase with an apparent Ki of 10 and 48 nM, respectively, as well as cause rapid time-dependent, first-order inactivation of aromatase in the presence of NADPH, whereas no inactivation was observed in the absence of NADPH. Substrate protects the enzyme from inactivation, but beta-mercaptoethanol does not, suggesting that the 6 beta-propargyl analogs 6 beta-PAD and 6 beta-PED are mechanism-based inactivators of aromatase. Energy-minimization calculations and molecular modeling studies indicate three global minima for each of the 6 beta-propargyl analogs in which one of the conformers is proposed to be responsible for the inactivation of aromatase.

In order to examine the nature of the hydrophobic pocket at the active site of aromatase, we carried out the synthesis, biochemical evaluation, and molecular modeling studies on 4-phenoxy-7 alpha-(phenylthio)-4-androstenedione 2. Aromatase inhibitory activity of 2 was found to be significantly weaker than that of the 4- and 7 alpha-mono(phenylthio)-substituted derivatives of androstenedione. These results along with those obtained from the modeling studies suggest the existence of a single hydrophobic pocket corresponding to the alpha-face in the C4, C6, C7 region of androstenedione.

The synthesis and biological evaluation of 4-thiosubstituted derivatives of 1,4-androstadienedione, 4,6-androstadienedione, and 1,4,6-androstatrienedione as inhibitors of aromatase are described. Inhibitory activity of synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H]androstenedione as substrate. Under initial velocity assay conditions of low product formation, the inhibitors demonstrated potent inhibition of aromatase, with apparent Kis ranging from 9.8 to 137 nM and with Km for androstenedione being 38 nM. However, unlike other 1,4-androstadienediones and 1,4,6-androstatrienediones in which time-dependent inactivation was observed, the 4-thiosubstituted analogs were found to be competitive inhibitors and did not produce any time-dependent inactivation of aromatase.

The DNA damage induced in a human breast cancer cell line treated with 1,5 (10)-estradiene-3,4,17-trione (3,4-estrone-o-quinone; 3,4-EQ) has been measured qualitatively and quantitatively. Single-strand (ss) but not double-strand (ds) DNA breaks were formed in MCF-7 cells treated with 3,4-EQ. The ss DNA breaks formed in MCF-7 cells were partially repaired after incubation of cells in 3,4-EQ-free media for 2 and 4 h (i.e. 33 and 23% repair, respectively, as compared to the ss DNA breaks in cells after a 1-h exposure to 3,4-EQ without a recovery period). The formation of interstrand DNA cross-links was demonstrated in MCF-7 cells exposed to the bifunctional alkylating agent, mitomycin C, but not in those exposed to 3,4-EQ. Protein-linked DNA breaks were detected in MCF-7 cells after exposure to camptothecin and etoposide but not 3,4-EQ, suggesting that the ss DNA breaks induced by 3,4-EQ are unlikely to be mediated via topoisomerases. The induction of ss DNA breaks was detected in the estrogen receptor-negative cell line, BT-20, after exposure to 3,4-EQ. Furthermore, excess estradiol in culture media did not prevent 3,4-EQ-induced ss DNA breaks, suggesting that the DNA damage was not mediated via the estrogen receptor. Evaluation of the newly synthesized quinone analogue, 5,6,7,8-tetrahydro-1-2-naphthoquinone, in the ss DNA breakage assay revealed that the A and B ring moiety of 3,4-EQ is sufficient to produce ss DNA breaks in MCF-7 cells.

Antipyrine (AP) is a commonly used probe of oxidative metabolism. Indirect evidence demonstrates formation rate limited disposition of its metabolites. Kinetic studies using antipyrine and its major metabolites 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA), and 4-hydroxyantipyrine (OHA) were completed to investigate the metabolic fate of preformed antipyrine metabolite and to demonstrate directly formation rate-limited metabolite disposition in vivo. Bolus injections of antipyrine and preformed metabolites (40-50 mg/kg) were administered to male, New Zealand white rabbits. Plasma and urine were analyzed using HPLC. These studies demonstrate that HMA, NORA, and OHA are formation rate limited in the rabbit. NORA appears to undergo further extensive oxidative and conjugative metabolism. Unknown additional peaks were detected in urine after NORA dosing but not after HMA or OHA administration. Mass spectroscopy of the unknown HPLC eluents identified potential structures of these NORA metabolites.

Microbial transformation of chromone, chromanone, and 3 ring A hydroxyflavones (5-hydroxy-, 6-hydroxy-, and 7-hydroxyflavones) was attempted using thirty-two microorganisms. While chromone was not biotransformed, chromanone was transformed to chromone and chromanol by Aspergillus niger in 2% yield. Ring A hydroxylated flavones were transformed to the corresponding C-4' hydroxylated metabolites, and the yields and rate of product formation were found to correlate with the distance between the C-4 carbonyl group and the hydroxyl group in ring A.

1. Flavone and isoflavone were subjected to microbial transformation studies. Screening with 80 fungal species showed that flavone was transformed by a large number of microorganisms and in higher yields than isoflavone. 2. Large-scale fermentations of flavone followed by isolation and characterization of metabolites using spectroscopic analysis showed the formation of 4'-hydroxyflavone, 3',4'-dihydroxyflavone and two cleavage products identified as o-hydroxyphenyl-hydroxymethyl ketone and 1-(o-hydroxyphenyl)-1,2-ethanediol. However, fermentation of isoflavone gave only 4'-hydroxyisoflavone and 3',4'-dihydroxyisoflavone.

The synthesis and evaluation of 4-thiosubstituted-4-androstenedione analogs as inhibitors of estrogen synthetase (aromatase) is described. All compounds were prepared by the addition of various thiol reagents to 4β,5β-epoxyandrostanedione. Inhibitory activity of synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1β- 3 H]4-androstene-3,17-dione as substrate. Synthesized compounds exhibiting high inhibitory activity were further evaluated under initial velocity conditions to determine apparent K i values. Several compounds were effective competitive inhibitors, and have apparent K i values ranging from 34 to 52 nM, with the apparent K m for androstenedione being 54 nM. The results of these studies demonstrate a tightly fitted enzyme pocket that can accommodate bulky substituents at the C-4 position of androstenedione not to exceed 4.3 A in width and 5.5 A in length.

The synthesis of 4-methylestradiol (4-ME2) was carried out by reductive aromatization of 4-methyl-1,4-androstadiene-3-one-17 beta-ol. The relative binding affinity of 4-ME2 was found to be 10 and 25% of estradiol at 0 and 25 degrees C, respectively. 4-ME2 had considerably weaker uterotrophic activity relative to estrone and was found to have no antiuterotrophic activity.

The synthesis and biological evaluation of 4-amino-, 4-alkoxy-, 4-aryloxy-, 4-alkyl- and 4-aryl-4-androstenedione derivatives as inhibitors of estrogen synthetase (aromatase) are described. Inhibitory activity of synthesized compounds was assessed using a human placental microsomal preparation as the enzyme source and [1 beta-3H] androstenedione as substrate. Synthesized compounds exhibiting aromatase inhibitory activity were evaluated further under initial velocity conditions to determine apparent Ki values. Several compounds were effective competitive inhibitors and have apparent Ki values ranging from 38 to 1290 nM, with the apparent Km for androstenedione being 47 nM. Alkylation or arylation of 4-N, S, or O-substituted steroids results in compounds that are effective competitive inhibitors that are devoid of time-dependent inactivation and that the free pair of electrons on N, S, or O is not an essential requirement for 4-substituted androstenedione derivatives to be effective aromatase inhibitors. The results obtained from this investigation are consistent with our previous studies which show that aromatase has a hydrophobic pocket in the active site around the C-4 alpha region of androstenedione.

The metabolism of the o-quinone derivative of estrone, 3,4-estrone quinone (3,4-EQ), has been investigated in human breast cancer cells. Unlike the p-quinone, diethylstilbestrol 4',4"-quinone, 3,4-EQ was not a substrate for the two-electron reduction catalyzed by the putative detoxifying enzyme, NAD(P)H:quinone reductase (DT diaphorase; DT D). Accordingly, the DNA damage induced by 3,4-EQ in human MCF-7 cells was not affected by an inhibitor of DT D. Although 3,4-EQ was not an apparent substrate for the two-electron reduction catalyzed by DT D, this o-quinone was a substrate for the one-electron reduction catalyzed by cytochrome P450 reductase. The one-electron reduction of 3,4-EQ catalyzed by cytochrome P450 reductase occurred in the face of a significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH. The impact of purified superoxide dismutase (SOD) upon the production of hydrogen peroxide produced as a consequence of 3,4-EQ metabolism was evaluated; surprisingly, SOD inhibited the hydrogen peroxide produced by this o-quinone. Possible reasons for the SOD-mediated inhibition of redox cycling of 3,4-EQ are discussed. In summary, important differences in the metabolism of 3,4-EQ vis-a-vis o- and p- quinones have been observed, and the implications of these differences are discussed.

The o-quinone forms of 2,3- and 3,4-catechol estrogens have been implicated in the carcinogenicity of these hormones. The concomitant production of reactive oxygen species during reduction of the o-quinone estrogens has been inferred to play a mechanistic role in their mutagenic potential. Conclusive evidence documenting the production of hydrogen peroxide, the hydroxyl radical, and the estrone 3,4-semiquinone in estrone 3,4-quinone (3,4-EQ)-treated human breast cancer subcellular fractions was demonstrated in the absence of exogenously added catalysts. Subcellular fractions of MCF-7 cells treated with 3,4-EQ and NADPH, including nuclei, mitochondria, and microsomes, were shown to support significant amounts of hydrogen peroxide production. Hydrogen peroxide production in 3,4-EQ-treated cellular fractions and the chromosomal DNA damage induced in 3,4-EQ-treated MCF-7 cells were abolished by the addition of catalase. A significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH resulting in hydrogen peroxide production was demonstrated. The results unequivocally demonstrate that free radicals are produced during the metabolism of estrone 3,4-quinone in human cells.

1. The synthesis and microbiological transformation of 2-phenyl-1-tetralone (compound 3, 1-deoxyisoflavanone), 3-phenyl-1-tetralone (compound 4, 1-deoxyflavanone), 2-phenylchroman (compound 7, 4-deoxyflavanone), 3-phenylchroman (compound 8, 4-deoxyisoflavanone) and 1,2-dihydro-3-phenylnaphthalene (compound 10, 1,4-dideoxy-dehydroflavanone) by Absidia blackesleeana are described. 2. Compounds 3, 4, 7 and 8 were hydroxylated at the 4'-position while compound 10 was not utilized as a substrate. The two phenylchroman analogues 7 and 8 gave approximately the same yield (22% and 26%, respectively) of the 4'-hydroxylation products, while the phenyltetralone analogues 3 and 4 showed significant differences in 4'-hydroxylation (2% and 47%, respectively).

Several synthetic flavones were found to inhibit the aromatization of androstenedione to estrone catalyzed by human placental microsomes. Twenty-one compounds were tested and the IC50 of the most active were: flavone, 10 microM; 7-hydroxyflavone, 0.5 microM; 7,4'-dihydroxyflavone, 2.0 microM; flavanone, 8.0 microM; and 4'-hydroxyflavanone, 10 microM. Most of the others had IC50 values ranging from 80 to greater than 200 microM. These findings show that 4'-hydroxylation results in either no change or very little change in IC50 for flavanone, isoflavone and isoflavanone as well as other ring A hydroxylated flavones. Derivatives of flavone with a hydroxyl substituent at position 5, 6 and 7 were also screened. 7-Hydroxyflavone (11) was the most effective competitive inhibitor (IC50 = 0.5 microM) with an apparent Ki value of 0.25 microM. Compound 11 also induced a change in the absorption spectrum of the aromatase cytochrome P-450 which is indicative of substrate displacement. The relative binding affinities of the flavonoid analogs were determined and only ring A adn ring B dihydroxylated analogs were found to bind to the estrogen receptor.

The glycoprotein cerato-ulmin is one of several toxic metabolites isolated from Ceratocystis ulmi , the causal agent of Dutch elm disease. We present a number of physico-chemical measurements on cerato-ulmin in water and in ethanol/water. Surface tension measurements in water reveal cerato-ulmin to be surface active, even at concentrations as low as 3×10 −8 g cm −3 . This property relates also to the change in drop si"2e observed as cerato-ulmin elutes during chromatography. Light scattering shows that cerato-ulmin is only sparingly soluble in 70% ethanol/water (3·7×10 −4 g cm −3 ) and much less soluble in water. A limited X-ray analysis provides an estimate of the molecular weight and some information about the aggregation of cerato-ulmin. Few resonances appear in the 13 C nuclear magnetic resonance (n.m.r.) spectrum for cerato-ulmin in water. However, more resonances appear in 75% ethanol/water, and many more in. 6 m urea. From these results we gain insight as to the intra- and intermolecular interactions of the macromolecule in these solvents. A possible mechanism for the toxic effect of cerato-ulmin at ultra-low concentrations is put forth.

The metabolism of estradiol was studied in 31 human breast carcinoma in vitro. All 16 estrogen-receptor-poor tumors transformed estradiol to estrone with percent conversions ranging from 11.4 to 95 except for one poorly differentiated tumor where 0.5% conversion to estrone was observed. On the contrary, only 3 out of 15 estrogen-receptor-rich tumors showed higher than 10% conversion of estradiol to estrone (p = 0.001). There is indication that the enzymatic activity in receptor-poor tumors steadily decreases in premenopausal patients as they approach menopausal age, whereas, the activity steadily increases in post-menopausal patients as the duration of menopause lengthens.

The inhibitory activity of aminoglutethimide, 4-hydroxy-androstenedione and 7α-(4'-amino) phenylthioandrostenedione on human placental and mammary tumor aromatase activity was examined. [14C]-Androstenedione was incubated with placental microsomes and mammary tumor homogenates in the presence of an NADPH generating system with or without inhibitor. All three inhibitors were equally effective in inhibiting microsomal placental aromatase at various inhibitor concentrations. Inhibitions of mammary tumor aromatase using 12 μM inhibitor concentrations were essentially similar to results on placental aromatase inhibition and ranged from 81–97% inhibition. These findings are discussed in regard to the potential clinical use of aromatase inhibitors in the treatment of estrogen dependent metastatic breast carcinoma.

An aggressive strain of Ceratocystis ulmi was grown in a defined medium for the production of phytotoxins. Antisera to two distinct toxins, concanavalin A binding glycopeptides and cerato-ulmin, were prepared and used as probes for the detection and localization of the toxins in toxin-treated cuttings of elm. The toxins caused wilting of elm cuttings and were detected primarily on the pit membranes of bordered pits.

[1 beta-3H], [1 alpha,2 alpha-3H] and [1 beta,2 beta-3H] 4-Hydroxyandrostenedione (4-OH-A) were synthesized to study the mechanism of inhibition of aromatase by 4-OH-A. Incubations of [1 beta-3H] and [1 beta,2 beta-3H] 4-OH-A with placental microsomes in the presence of NADPH showed very little loss of tritium, with aromatization of 4-OH-A ranging from 0.3 to 0.6 percent. No loss of tritium was observed in the absence of NADPH. The extent of covalent binding of 4-OH-A to microsomal proteins was higher with incubations in the absence of NADPH than with those in the presence of NADPH. These results are discussed in light of what has been proposed for the mechanism of androgen aromatization.

The glycoprotein cerato-ulmin, which shows surface activity at concentrations as low as 30 ng/mL, has been found to stabilize air bubbles, both spheroidal and of unusual morphology, in aqueous and aqueous ethanol solutions. In response to stress on a cerato-ulmin thin film, perhaps a monolayer, at an air–water interface, cylindrical and fibrilliform air bubbles are generated. They may be manipulated by application of tension, pressure, or temperature. The relationship of these observations to elm trees infected with the cerato-ulmin producing fungus Ceratocystis ulmi suggests a mechanism whereby exceedingly low concentrations of cerato-ulmin may be concentrated and delivered to intercellular openings, thereby blocking the vascular system of elm trees. The correctness of this mechanism as a major factor in any tree wilt disease remains to be tested.

Twenty-two algal cultures grown photoautotrophically were incubated with 4-androstene-3,17-dione and 17 beta-hydroxy-4-androstene-3-one. Most cultures were capable of effecting transformation. The metabolites were isolated and detected by tlc and hplc, and were identified by comparison of chromatographic and/or spectral properties with those of authentic compounds. Most cultures showed the presence of 17-oxidoreductase, some were capable of hydroxylating 4-androstenedione at the 6 beta- and 14 alpha-positions, and a few were capable of utilizing substrates as an energy and/or a carbon source. Incubations with testosterone showed very little metabolism.

A 5β-H steroid binding component using etiocholanolone has been identified in chick embryo liver which exhibits characteristics of a specific receptor. Kinetic analysis revealed the presence of specific binding sites with a Kd of 8.9 × 10−10 M and a concentration of binding sites of 59 fmol/mg protein. The nuclear and cytosol macromolecule has a sedimentation coefficient of 4.6 under low and high salt conditions. Following in vivo injection of etiocholanolone to chick embryos or in vitro preincubation of chick embryo tissue slices at 30°C the cytosol binding protein was markedly reduced while the nuclear binding protein increased. However, etiocholanolone preincubation of tissue slices at 0°C resulted in no receptor translocation. Competition studies using a 200-fold excess of various unlabelled steroids demonstrated greatest inhibition with steroids having the 5β-H configuration. These data offer strong evidence for the presence of a 5β-H steroid receptor in chick embryo liver.

The synthesis and evaluation of 4-(substituted thio)-4-androstene-3,17-dione derivatives as inhibitors of estrogen synthetase (aromatase) are described. All compounds were prepared by the addition of various thiol reagents to 4 beta,5 beta-epoxyandrostanedione. Inhibitory activity of synthesized compounds was determined with use of a human placental microsomal preparation as the enzyme source and [1 beta-3H]-4-androstene-3,17-dione as substrate. Synthesized compounds exhibiting high inhibitory activity were further evaluated under initial velocity conditions to determine apparent Ki values. Several compounds were effective competitive inhibitors and have apparent Ki values ranging from 36 to 73 nM, with the apparent Km for androstenedione being 53 nM. The results of these studies demonstrate a tightly fitted enzyme pocket that can accommodate bulk up to about 5.5 A.

The activities of estrogen-2,4- and 16 alpha-hydroxylase as well as 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSDHase) were determined in normal, benign and mammary tumor breast tissues. All three hydroxylases were absent in both normal and benign tumor breast tissues. 17 beta-OHSDHase was absent from normal breast tissues but present in all benign tumor tissues. Estrogen-2-hydroxylase was present in most breast carcinomas, was significantly higher in estrogen receptor (ER)-positive than ER-negative tumors, did not correlate with progesterone receptor (PR) and was significantly lower in the ER+PR- subgroup of breast cancers. 4-Hydroxylase activity did not correlate with either ER or PR content but was significantly lower in the ER+PR- subgroup of breast cancers. The activity of 16 alpha-hydroxylase was present in only 18% of all tumors investigated (9/50), did not correlate with the ER or PR content and was completely absent in the ER+PR- and ER-PR+ subgroups. The activity of 17 beta-OHSDHase was significantly higher than the estrogen hydroxylase but did not correlate with either the ER or PR content, and was not different among the ERPR tumor subgroups. The physiological role of these enzymes in the metabolism of estradiol in relation to breast cancer is discussed.

Reaction of estrone-3,4-o-quinone with ethanethiol and glutathione leads to the formation of 4-hydroxyestrone-2-thioethers. Incubations of [1-3H]hydroxyestrone with rat liver microsomes and NADPH in the presence of glutathione results in the formation of 4-hydroxyestrone-S-glutathione with no release of tritium in the water indicating GSH addition to C-2 of 4-hydroxyestrone.

1. Zearalenone, a fungal oestrogenic compound, was subjected to microbial transformation studies. Preliminary screening with 150 fungal species showed that Gliocladium roseum was capable of metabolizing zearalenone in 80-90% yields. 2. Large-scale fermentation with G. roseum produced a 1:1 mixture of 1-(3,5-dihydroxyphenyl)-10'-hydroxy-1-undecen-6'-one and 1-(3,5-dihydroxyphenyl)-6'-hydroxy-1-undecen-10'-one. The compounds were isolated and purified at -20 degrees C, and identified using spectroscopic analysis and by comparison to products obtained from alkaline hydrolysis of zearalenone.

The conversion of 5-hydroxyflavone by various microorganisms was studied. Among them, Streptomyces fulvissimus was the sole microbe which produced a new polar metabolite from 5-hydroxyflavone in addition to 5,4-dihydoxy- and 5,3,4-trihydroxyflavone. The structure of this polar metabolite was determined to be 5,4-dihydroxyflavone-4-sulfate on the basis of mass, infrared, and nuclear magnetic resonance spectroscopies. These results demonstrate that S. fulvissimus catalyzes sulfation at the 4 position of 4,5-dihydroxyflavone.

The synthesis and evaluation of 4-(substituted thio)-4-androstene-3,17-dione derivatives as inhibitors of estrogen synthetase (aromatase) are described. All compounds were prepared by the addition of various thiol reagents to 4 beta,5 beta-epoxyandrostanedione. Inhibitory activity of synthesized compounds was determined with use of a human placental microsomal preparation as the enzyme source and [1 beta-3H]-4-androstene-3,17-dione as substrate. Synthesized compounds exhibiting high inhibitory activity were further evaluated under initial velocity conditions to determine apparent Ki values. Several compounds were effective competitive inhibitors and have apparent Ki values ranging from 36 to 73 nM, with the apparent Km for androstenedione being 53 nM. The results of these studies demonstrate a tightly fitted enzyme pocket that can accommodate bulk up to about 5.5 A.

Ponasterone A(I) is converted by the microorganism Fusarium lini ATCC 9593 into rubrosterone (IV) in 15% yield.

The effect of ascorbic acid on the growth of 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumor and on growth of R3230AC and MT/W9a-B transplantable rat mammary tumors was investigated. High doses of ascorbic acid averaging about 540 mg/day/rat administered orally in drinking water had no effect on the growth of both R3230A and MT/W9a-B transplantable mammary tumors. Furthermore, vitamin C was unable to postpone tumor induction, reduce tumor incidence or prolong survival time of rats treated with DMBA.

The binding of tritiated etiocholanolone to rat liver and Novikoff hepatoma cells has been investigated. Cytosols obtained from rat liver and Novikoff hepatoma contain high affinity-low capacity binding macromolecules (Kd = 9.6 × 10−10 M for rat liver and 1.1 × 10−9 M for Novikoff hepatoma and the number of binding sites was 64 fmol/mg protein for rat liver and 76 fmol/mg protein for Novikoff hepatoma). Cytosols labelled with [3H]-etiocholanolone had a sedimentation coefficient of 4.6 under low and high salt concentration. Preincubation of rat liver slices and Novikoff hepatoma cells with unlabelled etiocholanolone followed by exchange of nuclear receptor complex with [3H]-etiocholanolone showed the presence of a single peak sedimenting at 4.6 S. Extraction of nuclear receptor peak sedimenting at 4.6 S and identification of the steroid showed that 86% of the radioactivity was associated with etiocholanolone and 14% with 5β-androstane-3α,17β-diol. Competition studies showed that steroids with 5β-H configuration were very effective in displacing etiocholanolone from cytoplasmic receptors while steroids with the 5α-configuration were not. These studies show that etiocholanolone receptor present in mammalian liver is essentially similar to that obtained from chick embryo liver.

Short ragweed leaves stems, roots, and seeds were examined for the presence of the most potent pollen allergen, antigen E. Soluble proteins were precipitated by ammonium sulfate and partially purified by a combination of dialysis and anion exchange and gel filtration chromatography. Immunodiffusion, disk electrophoresis, and skin testing of short ragweed pollen-sensitive individuals indicated that antigen E was present in leaves-stems portion of this plant as well as the pollen.

Dehydrogenation of 5α-3-ketosteroids with cell-free preparations of Nocardia restrictus involves a cis removal of the 4α and 5α hydrogens while dehydrogenation of 5β-3-ketosteroids proceeds by a stereospecific removal of the 4α and 5β hydrogens.

Ponasterone A(I) is converted by the microorganism Fusarium lini ATCC 9593 into rubrosterone (IV) in 15% yield.