James C. Warren

Effects of estrogen on glucose tolerance

Abstract Fourteen women were treated with diethylstilbestrol or norethynodrel with mestranol for 30 days. The oral and intravenous glucose tolerance test was done before and after treatment. Eleven patients had a diabetic oral glucose tolerance curve following treatment. Because the intravenous glucose tolerance curves were normal, it is suggested that the diabetic oral curves may be due to delayed gastrointestinal absorption, although direct data on this point are not available. Patients who display diabetic oral glucose tolerance curves while taking estrogens should be reexamined either with the intravenous test or after discontinuance of estrogen therapy.

An estrophilic macromolecula in chicken liver cytosol

Abstract 1. 1. The existence of a 4-S estrophilic macromolecule in chicken liver cytosol has been demonstrated by sucrose density gradient analysis of cytosol fractions incubated with [6,7- 3 H 2 ]estradiol-17β and similar analysis of cytosol fractions after intravenous adminstration of the tritiated steroid to laying hens and castrated male chickens. The estrophile binds estradiol-17β but not progesterone or testosterone. The K d estradiol-17β approximates 2·10 −9 M. The binding is destroyed by treatment with pronase, but unchanged by treatment with ribonuclease. 2. 2. The role of this macromolecule in the nuclear transport of estradiol was studied by comparing nuclear preparations incubated wity cytosol containing estrophile- bound estradiol with similar preparations incubated with Tris-EDTA buffer containing free estradiol. In this cell-free system, the estrophile retards total transfer of tritiated steroid into the nucleus. Nevertheless, the tritiated steroid extracted with 0.4 M KCl from nuclei after incubation with estrophile-bound estradiol is clearly of a different character in that: (a) it is mostly bound to a macromolecule excluded by Sephadex G-25 and G-50; and (b) it is primarily still estradiol. That extracted with 0.4 M KCl from nuclei after incubation with Tris-EDTA containing estradiol is (a) mostly free; and (b) over 50% converted to estrone.

Reaction mechanism and stereospecificity of 20 β-hydroxysteroid dehydrogenase ☆

Abstract Kinetic and spectral analyses of 20β-hydroxysteroid dehydrogenase indicate that this enzyme mediates an Ordered BiBi reaction with cofactor binding first. Dissociation constants of NAD + and NADH have been determined by initial velocity studies and show that the enzyme has a higher affinity (10 to 15-fold) for NADH than for NAD + . The enzyme mediates hydrogen transfer from the 20α-position of the steroid to the 4β-position of the cofactor pyridine ring. Kinetic analysis of site specificity using pregnenolone and its sulfate ester indicate that both compounds are reduced at the same enzymatic site.

Artificial Insemination with Fresh Donor Semen

A total of 141 patients received donor insemination during a 21/2-year period. Pregnancy was achieved by 57 per cent of these patients. When patients who were inseminated for less than three cycles are excluded, the pregnancy rate is 77 per cent. The mean conception time was three treatment cycles. In 69 pregnancies, the occurrence of abortion was that of the general population, and an increased male-to-female ratio was observed in the 36 live-born infants. Questionnaires sent to all couples and returned by 81 per cent indicated that donor insemination was not emotionally harmful to their marriage. Heterologous insemination is a safe, effective therapeutic alternative to adoption for selected couples.

Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.

Purification and properties of 3-beta-hydroxysteroid dehydrogenase and delta-5-3-ketosteroid isomerase from bovine corpora lutea.

Abstract 1. 1. Properties and kinetics of 3β- Hydroxysteroid dehydrogenase and Δ 5 -3- ketosteroid isomerase from the bovine ovary have been examined. The enzymes were purified from the microsomal fraction utilizing sonication, (NH4)2SO4 precipitation, dialysis, and column chromatography. With DPN+ as cofactor, the purified dehydrogenase oxidizes the 3β- hydroxy position of C19 and C20 steroids of the Δ 4 , Δ 5 , and 5α series. It was inactive with 3α-, 11β-, 17β- and 21- hydroxy positions as well as the 3β- position of cholesterol and did not utilize TPN+ as cofactor. 2. 2. With a Δ 5 -3β- hydroxy compound as substrate there was no significant formation of the Δ 4 -3β- hydroxy compound during incubation without nucleotide cofactor. Thus, the reactions leading to Δ 4 -3- ketones appear to be ordered, with oxidation of Δ 5 -3β- hydroxy compounds to Δ 5 -3- ketones preceding isomerization to Δ 4 -3- ketones . 3. 3. Behaviour during purification indicated that failure to obtain complete solubilization is responsible for lack of success in complete separation of dehydrogenase and isomerase activities. However, non-identity of the sites effecting dehydrogenation and isomerization is indicated by variation of activity ratios during purification, different inactivation rates and different pH curves. 4. 4. The presence of a single dehydrogenase site for C19 and C21 substrates and a single isomerase site for C19 and C20 substrate is indicated by the relative constancy in activity ratios during purification, the kinetics of equimolar mixtures, the competitive activation by alternate substrates, and the similarities of inactivation rates.

Site specificity and mechanism of human placental 17β-hydroxysteroid dehydrogenase

Abstract Kinetic analysis of the site specificity of purified human placental 17β-hydroxysteroid dehydrogenase in terms of cofactor (NAD + and NADP + ) and steroid (estradiol-17β and estradiol 3-sulfate) indicate that estradiol-17β and estradiol 3-sulfate bind at a single activ-site. While NAD + and NADP + have an active site in common, a second NAD + binding site (active or allosteric) of lower binding affinity is also present. Product inhibition studies are compatible with an Ordered Bi Bi mechanism with steroid binding first or an Iso Theorell-Chance mechanism with cofactor binding first.

Sulfatase activity in the human placenta

Abstract Sulfatase(s) capable of hydrolyzing the 3-sulfates of dehydroepiandrosterone, estrone and cholesterol and the sulfate ester of p-nitrophenol are present in human placenta, wholly or almost exclusively in the microsomal fraction. Placenta is incapable of effecting detectable hydrolysis of testosterone-17β-sulfate and the 16α-sulfate of 16α-hydroxyprogesterone.

Site specificity of bovine adrenal 3β-hydroxysteroid dehydrogenase and δ5-3-ketosteroid isomerase

Abstract 1. 1. The 3β-hydroxysteroid dehydrogenase and Δ 5 -3-ketosteroid isomerase activities from the microsomal fraction of bovine adrenal cortex have been studied with emphasis on the site specificity for dehydrogenation and isomerization of the natural C 19 and C 21 steroid substrates. 2. 2. The data indicate a distinct dehydrogenase site and a distinct isomerase site each of which is capable of utilizing both C 19 and C 21 substrates as shown by the following parameters: activity ratios during purification, pH curves, inactivation rates, and the kinetics of equimolar mixtures. These observations are similar to results with the same activities from bovine corpora lutea.

Interceptives: drugs interrupting pregnancy after implantation.

The mechanism and efficacy of Oxymetholone and Durabolin for interrupting pregnancy were studied in 56 pregnant Holtzman rats. Each drug was administered individually to separate groups by subcutaneous injection for 6 days starting on the 7th day of pregnancy. Phenobarbital was administered to a third group of rats and a fourth untreated group were used as controls. 96% of the controls bore normal fetuses while the phenobarbital group showed no significant alteration of their pregnancy rate. 5 mg of Oxymetholone daily caused resorption of pregnancy in 87.5% of the animals with very small traces of remaining placentae. 2 mg of Durabolin daily caused 87.5% resorption of pregnancy while higher doses achieved 100% resorption with significant remains of placentae. When 5 mg of progesterone was administered simultaneously with the 5 mg of Oxymetholone a normal number (87.5%) of pregnancies occurred. When the Durabolin dose was reduced to 1 mg daily the 5 mg dose of progesterone accompanying it also returned the pregnancy rate to normal. Oxymetholone appears to act by suppressing gonadotropins. The action of Durabolin has yet to be determined; its mechanism is not catatoxic or antigonadotropic but probably interferes with progesterone synthesis. Further research is indicated for these interceptives of pregnancy which are active after implantation and could be used as a means of birth control.