UDP-N-acetyl-D-galactosamine as a donor substrate for the glycosyltransferase encoded by the B gene at the human blood group ABO locus

The properties of the enzyme in the serum of blood group B individuals that catalyses the transfer of small amounts of N-acetyl-D-galactosamine to H-active precursor structures were compared with those of the blood group B gene-associated alpha-(1----3)-D-galactosyltransferase and with the blood group A gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases in the serum of blood group A1 and A2 individuals. The biosynthetic products formed by the enzyme in B serum were identical with the A-active structures synthesised by the A1 and A2 gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases but the enzyme differed from the A1 and A2 transferases in its apparent Km for UDP-N-acetyl-D-galactosamine, its heat susceptibility, its failure to bind to Sepharose 4B, and its adsorption to H-active sites on group O red cell ghosts under conditions which bind the B transferase but fail to adsorb the A1 and A2 transferases. The correlation between the levels of alpha-(1----3)-D-galactosyltransferase and alpha-(1----3)-N-acetyl-D-galactosaminyltransferase activities in all the group B serum samples tested, the maintenance of the same ratio of activities after successive cycles of binding to group O red cell ghosts, the retention of the ability to convert blood group O to A-active cells after treatment of the serum with Sepharose 4B, and the failure to detect any comparable activity in group O serum samples tested under the same conditions indicated that the enzyme in group B serum that utilises UDP-N-acetyl-D-galactosamine to make blood group A-active structures is the B gene-associated alpha-(1----3)-D-galactosyltransferase.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.     

Association of the human Lewis blood group Le(a-b-c-d-) with the failure of expression of alpha-3-L fucosyltransferase

Two unrelated individuals are reported who lack alpha-3-L-fucosyltransferase activity in their serum and saliva. Both were blacks, one from the United States and the other from South Africa. No other of the tested members of their families lacked this enzyme. A survey of more than 2000 serum samples from both black and white South African blood donors, black United States donors and white United Kingdom donors failed to disclose another example of a serum deficient in alpha-3-L-fucosyltransferase activity. The two individuals lacking in alpha-3-L-fucosyltransferase activity both had the Lewis blood group phenotype Le(a-b-c-d-). No other persons with this phenotype have been reported. The absence of Lec activity in the two individuals who are deficient in alpha-3-L-fucosyltransferase is consistent with the interpretation that alpha-3-linked L-fucose is an essential part of the antigenic determinant recognised by the anti-Lec reagent used in this investigation.     

Acceptor specificity and tissue distribution of three human alpha-3-fucosyltransferases

Based on the capacity to transfer alpha-L-fucose onto type-1 and type-2 synthetic blood group H and sialylated acceptors, a comparison of the alpha-3-fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid alpha-3-fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type-2 acceptor and poorly onto sialylated N-acetyllactosamine: (II) plasma alpha-3-fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N-acetyllactosamine; (III) Lewis alpha-3 4-fucosyltransferase (EC 2.4.1.65), in which gall-bladder kidney and milk enzymes transfer, in addition, onto type-1 acceptors. The small amount (less than 10%) of alpha-3-fucosyltransferase activity found in the plasma of an alpha-3-fucosyltransferase-deficient individual had a myeloid-type acceptor pattern, suggesting that this small proportion of the plasma enzyme is derived from leukocytes. In addition to the three acceptor specificity patterns, these enzyme activities can be differentiated by their optimum pH: 8.0-8.7 for the enzymes from myeloid cells and brain. 7.2-8.0 for liver enzymes and 6.0-7.2 for gallbladder enzymes. Milk samples had two alpha-3-fucosyltransferase activities, the Lewis or alpha-3/4-fucosyltransferase under control of the Lewis gene and an alpha-3-fucosyltransferase with plasma acceptor pattern which was independent of the control of the Lewis gene. The apparent affinity for GDP-fucose of the myeloid-like enzyme was weaker than those of the plasma and Lewis-like enzymes. The apparent affinities for H type 2 and sialylated N-acetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of alpha-3-fucosyltransferase activity was more sensitive to N-ethylmaleimide and heat inactivation than the samples with myeloid-like alpha-3-fucosyltransferase activity.     

Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens

Poly-N-acetyllactosamines (pLNs) are common terminal sugars of many N- and O-linked glycan structures present in glycoproteins and glycolipids. Utilizing various glycosyltransferases, we developed new and efficient chemoenzymatic methods for the synthesis of pLNs in gram-scale. Specifically, the use of sialyltransferases and fucosyltransferases enabled us to synthesize and purify 24 blood group and tumor-associated pLN derivatives with alpha-(2-->3)- and alpha-(2-->6)-linked sialic acid, as well as with alpha-(1-->2)- and alpha-(1-->3)-linked fucose. All synthesized derivatives were linked to a short 2-azidoethyl spacer for further modification.     

Interactions of the bovine brain A1-adenosine receptor with recombinant G protein alpha-subunits. Selectivity for rGi alpha-3

The ability of the bovine brain A1-adenosine receptor to discriminate between different G protein subtypes was tested using G protein alpha-subunits synthesized in Escherichia coli (rG alpha-subunits). When combined with a 3-fold molar excess of beta gamma-subunit purified from bovine brain and used at high concentrations, all three subtypes of rGi alpha (rGi alpha-1, rGi alpha-2, and rGi alpha-3) and rGo alpha were capable of reconstituting guanine nucleotide-sensitive high-affinity binding of the agonist radioligand (-)-N6-3-[125I] (iodo-4-hydroxyphenylisopropyl) adenosine ([125I]HPIA) to the purified A1-adenosine receptor (Kd approximately 1.2 nM). Titration of the A1-adenosine receptor with increasing amounts of rG alpha revealed a approximately 10-fold higher affinity for rGi alpha-3 compared with rGi alpha-1, rGi alpha-2, and rGo alpha. This selectivity was also observed in the absence of beta gamma. Other alpha-subunits (rGs alpha-s, rGs alpha-L, rGs alpha PT, and rGz alpha) did not promote [125I]HPIA binding to the purified receptor. In N-ethylmaleimide-treated bovine brain membranes, rGi alpha-3 was the only rG alpha-subunit capable of reconstituting high-affinity agonist binding. Similarly, rGi alpha-3 competed potently with rGo alpha for activation by the agonist-liganded A1-adenosine receptor, whereas a approximately 50-fold molar excess of rGo alpha was required to quench the receptor-mediated release of [alpha-32P]GDP from rGi alpha-3. Hence, in spite of the extensive homology between alpha-subunits belonging to the Gi/Go group, the A1-adenosine receptor appears to discriminate between the subtypes. This specificity is likely to govern transmembrane signaling pathways in vivo.     

Synthesis of specifically deuterium-labelled pregnanolone and pregnanediol sulphates for metabolic studies in humans.

A synthesis is reported of 3beta-hydroxy-5alpha-pregnan-20-one sulphate and the disulphate and 3-monosulphate of 5alpha-pregnane-3beta,20alpha-diol, labelled specifically with deuterium in high isotopic purity for metabolic studies in humans. Base-catalyzed equilibration of 3beta-hydroxy-5alpha-25R-spirostan-12-one (hemcogenin, II) with deuterium oxide, followed by removal of the 12-keto group and degradation of the sapogenin side-chain afforded 3beta-hydroxy-5alpha-[11,11-2H2]pregn-16-en-20-one (VII). Further deuterium atoms were introduced at the 3alpha and 20beta positions by reductions with sodium borodeuteride and lithium aluminum deuteride, respectively. These reactions led to 3beta-hydroxy-5alpha-[3alpha,11,11-2H3]pregnan-20-one (X; isotopic purity 87.2%) and 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol (XIV; isotopic purity 83.9%). The 3-sulphate of the pregnanolone and the 3,20-disulphate of the pregnanediol were prepared directly form the free alcohols, while the 3-monosulphate of the pregnanediol was obtained via 5alpha-[3alpha,11,11,20beta-2H4]pregnane-3beta,20alpha-diol 20-acetate (XVII).     

Synthesis of 2-[(2-pyridyl)amino]ethyl beta-D-lactosaminide and evaluation of its acceptor ability for sialyltransferase: a comparison with 4-methylumbelliferyl and dansyl beta-D-lactosaminide

We reported the synthesis of beta-D-lactosaminide with a 2-aminopyridyl group that is linked to a glycosyl tether at the reducing end. This fluorescent disaccharide acts as an acceptor for both alpha-(2-->6)- and alpha-(2-->3)-sialyltransferases. In addition, the acceptor ability of this disaccharide was evaluated and compared with that of beta-D-lactosaminide having a dansyl or a 4-methylumbelliferyl group.     

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.     

Preservation of Alpha-3 Neuronal Nicotinic Acetylcholine Receptor Expression in Sympathetic Ganglia After Brain Death

The goal of this study was to evaluate if the immunohistochemical expression of alpha-3 neuronal nicotinic acetylcholine receptor subunit in sympathetic ganglia remains stable after brain death, determining the possible use of sympathetic thoracic ganglia from subjects after brain death as study group. The third left sympathetic ganglion was resected from patients divided in two groups: BD—organ donors after brain death and CON—patients submitted to sympathectomy for hyperhidrosis (control group). Immunohistochemical staining for alpha-3 neuronal nicotinic acetylcholine receptor subunit was performed; strong and weak expression areas were quantified in both groups. The BD group showed strong alpha-3 neuronal nicotinic acetylcholine receptor expression in 6.55% of the total area, whereas the CON group showed strong expression in 5.91% (p = 0.78). Weak expression was found in 6.47% of brain-dead subjects and in 7.23% of control subjects (p = 0.31). Brain death did not affect the results of the immunohistochemical analysis of sympathetic ganglia, and its use as study group is feasible.     

Two new alkaloidal taxoids from the needles of Taxus canadensis

Two new taxanes with a dimethylamino group on the C-5 side chain were identified for the first time in the needles of the Canadian yew, Taxus canadensis. Their structures were characterized as 7beta,10beta,13alpha-triacetoxy-5alpha-(3'-dimethylamino-3'-phenylpropanoyl)oxy-2alpha-hydroxy-2(3-->20)abeotaxa-4(20),11-dien-9-one (1) and 2alpha,10beta-diacetoxy-9alpha-hydroxy-5alpha-(3'-dimethylamino-3'-phenylpropanoyl)oxy-3,11-cyclotax-4(20)-en-13-one (2).     

Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins.

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAc alpha-], BSA-[Gal(beta 1-3)GalNAc(alpha-], and BSA-[Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc(alpha-], were prepared. alpha GalNAc derivatives were galactosylated employing crude beta-galactosidase from bovine testes. The use of oversaturated donor solutions (pNP beta Gal) enhanced the yields up to 60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated products. Further treatment of the disaccharides with CMP-Neu5Ac and alpha 2-3 sialyltransferase from pork liver led to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide components. The results were monitored by HPLC and MALDI-TOF.     

Synthesis of fluorescently labelled and internally quenched UDP-Gal probes

The preparation of fluorescently labelled and internally quenched UDP-Gal probes bearing a fluorescence emitter and a quencher is described. The rate of transfer using several galactosyltransferases was examined. Our results demonstrate that galactose-modified, sugar-nucleotide-modified and double modified UDP-Gal analogues are recognized as weak substrates by blood group B alpha-(1-->3) galactosyltransferase, alpha-(1-->3) galactosyltransferase and milk bovine beta-(1-->4) galactosyltransferase.     

Biosynthesis of disialylated beta-D-galactopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopy ran osyl oligosaccharide chains. Identification of a beta-D-galactoside alpha-(2----3)- and a 2-acetamido-2-deoxy-beta-D-glucoside alpha-(2----6)-sialyltransferase in regenerating rat liver and other tissues

Regenerating rat liver microsomes contain a beta-D-galactoside alpha-(2----3)- and a 2-acetamido-2-deoxy-beta-D-glucoside alpha-(2----6)-sialyltransferase that are involved in the synthesis of the terminal alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-alpha-[NeuAc-(2----6)]-beta- D-GlcpNAc-(1----R) group occurring in human milk oligosaccharides and the glycan chains of several N-glycoproteins. Analysis by liquid chromatography and methylation of the products of sialylation obtained when lacto-N-tetraose [beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4) -D-Glc] was used as a substrate in the incubations in vitro indicated that the disialylated sequence is formed for greater than 95% through the tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----3)-beta-D-G al- (1----4)-D-Glc as one of two possible intermediates. This indicates that in the synthesis of the disialylated sequence the alpha-(2----3)- and the alpha-(2----6)-sialyltransferase act in a highly preferred order in which the alpha-(2----3) enzyme acts first. This order is imposed by the specificity of the alpha-(2----6)-sialyltransferase, which requires an alpha-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----R) sequence for optimal activity, and shows very low and no activity with beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----R) and beta-D-GlcNAc-(1----R) acceptor structures, respectively. Results obtained with normal rat, fetal calf, rabbit and human liver, and human placenta indicated that very similar or identical sialyltransferases occur in these tissues. It is suggested that these enzymes differ from the sialyltransferases that previously had been identified in fetal calf liver and human placenta.     

Glycoinositolphosphosphingolipids (basidiolipids) of higher mushrooms.

The basidiolipids of six mushroom species, i.e. the basidiomycetes Amanita virosa (engl., death cup), Calvatia exipuliformis (engl., puffball), Cantharellus cibarius (engl., chanterelle), Leccinum scabrum (engl., red birch boletus), Lentinus edodes (jap., Shiitake), and Pleurotus ostreatus (engl., oystermushroom), were isolated, and their chemical structures investigated. All glycolipids are structurally related to those of the Agaricales (engl., field mushroom). They are glycoinositolphosphosphingolipids, their ceramide moiety consisting of t18:0-trihydroxysphinganine and an alpha-hydroxy long-chain fatty acid. In contrast to a previous study [Jennemann, R., Bauer, B.L., Bertalanffy, H., Geyer, R., Gschwind, R.M., Selmer, T. & Wiegandt, H. (1999) Eur. J. Biochem. 259, 331--338], the glycoside anomery of the hexose (mannose) connected to the inositol of all investigated basidiomycete glycolipids, including the basidiolipids of Agaricus bisporus, was determined unequivocally to be alpha. Therefore, the root structure of all basidiolipids consists of alpha-DManp-2Ins1-[PO(4)]-Cer. In addition, for some mushroom species, the occurrence of an inositol substitution position variant, alpha-Manp-4Ins1-[PO(40]-Cer, is shown. The carbohydrate of chanterelle basidiolipids consists solely of mannose, i.e. Cc1, Man alpha-3 or -6Man alpha; Cc2, Man alpha-3(Man alpha-6)Man alpha-. All other species investigated show extension of the alpha-mannoside in the 6-position by beta-galactoside, which, in some instances, is alpha-fucosylated in 2-position (Fuc alpha-2)Gal beta-6Man alpha-. Further sugar chain elongation at the beta-galactoside may be in 3- and/or 6-position by alpha-galactoside, e.g. Ce4, Po2, Gal alpha-3-(Gal alpha-6)(Fuc alpha-2)Gal beta-6Man alpha-, whereas A. virosa, Av-3, has a more complex, highly alpha-fucosylated terminus, Gal alpha-3 (Fuc alpha-2)(Fuc alpha-6)Gal alpha-2(Gal alpha-3)Gal beta-6Man alpha-. L. edodes basidiolipids show further elongation by alpha-mannoside, e.g. Le3, Man alpha-2Man alpha-6Gal alpha-3(Fuc alpha-2)Gal beta-6Man alpha-, C. exipuliformis glycolipid by alpha-glucoside, i.e. Ce3, Glc alpha-6Gal beta-6Man alpha-. Basidiolipid Ls1 from L. scabrum, notably, has a 3-alpha-mannosylated alpha-fucose, i.e. Gal alpha-6(Man alpha-3Fuc alpha-2)Gal alpha-6Gal beta-6Man alpha-. In conclusion, basidiolipids, though identical in their ceramide constitution, display wide and systematic mushroom species dependent variabilities of their chemical structures.     

alpha-DNA II. Synthesis of unnatural alpha-anomeric oligodeoxyribonucleotides containing the four usual bases and study of their substrate activities for nucleases.

This paper describes for the first time the synthesis of alpha-oligonucleotides containing the four usual bases. Two unnatural hexadeoxyribonucleotides: alpha-[d(CpApTpGpCpG)] and alpha-[d(CpGpCpApTpG)], consisting only of alpha-anomeric nucleotide units, were obtained by an improved phosphotriester method, in solution. Starting material was the four base-protected alpha-deoxyribonucleosides 3a-d. Pyrimidine alpha-deoxynucleosides 3a and 3b were prepared by self-anomerization reactions followed by selective deprotection of sugar hydroxyles, while the two purine alpha-deoxynucleosides 3c and 3d were prepared by glycosylation reactions. In the case of guanine alpha-nucleoside derivative a supplementary base-protecting group: N,N-diphenylcarbamoyl was introduced on O6-position in order to avoid side-reactions during oligonucleotide assembling. The hexadeoxynucleotide alpha-[d(CpApTpGpCpG)] was tested as substrate of selected endo- and exonucleases. In conditions where the natural corresponding beta-hexamer was completely degradated by nuclease S1 and calf spleen phosphodiesterase, the alpha-oligonucleotide remained almost intact.     

Effect of dietary cyclic nigerosylnigerose on intestinal immune functions in mice

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.     

Conversion of 17 alpha-hydroxyprogesterone to 5 alpha, 3 alpha, and 20 alpha-reduced metabolites by female rat anterior pituitary and hypothalamus

The metabolism of 17 alpha-[3H]hydroxyprogesterone was examined in female rat anterior pituitary and hypothalamic tissues. After reverse isotopic dilution analysis and purification to constant specific activity, the following 5 alpha-, 3 alpha- and 20 alpha-reduced products were detected in both tissues: 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione; 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol. While the metabolites formed were qualitatively the same, there were quantitative differences between the two tissues. The 3 alpha,5 alpha-reduced metabolite, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one, was the principal product in the anterior pituitary while the 5 alpha-reduced metabolite, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, was produced in largest amount by the hypothalamus. With both tissues, the aforementioned four products plus starting substrate accounted for nearly all of the starting radioactivity. There was no evidence for the formation of C19 steroids (androgens) despite the presence of the 17 alpha-hydroxy group.