John S. Schweppe

In vitro synthesis of estrogens by midterm human placenta and fetal liver.

Abstract Radioactive progesterone, 17α-hydroxypregnenolone, androstenedione and testosterone were incubated with midterm human fetal liver and placenta. After isolation of the radioactive phenolic metabolites, purification was achieved by paper chromatography and crystallization to constant specific activity. Fetal liver converted androstenedione and testosterone to estrone, 17β-estradiol and estriol. Estriol, but no estrone and 17β-estradiol could be identified from the incubation of progesterone and 17α-hydroxy-pregnenolone. The corresponding midterm placenta readily aromatized androstenedione and testosterone to estrone, 17β-estradiol and estriol. The conversion of progesterone and 17α-hydroxypregnenolone to estrogens was small and varied between 0.2 and 0.34%.

Charge heterogeneity of human low density lipoprotein (LDL)

Summary Plasma samples were obtained from patients with known history of atherosclerosis as well as from a group of Bantus of South Africa and a group of medical students without any history of atherosclerotic vascular disease. The isoelectric point (IEP) of LDL of the different persons was determined by electrophoresis in agarose gel at different pH values. The IEP of the LDL of the students or of the Bantus, were fairly constant at pH 5.28; for the patients the IEP varied widely. The IEP of LDL from hypercholesteremic persons (Fredrickson type II) showed consistently a significantly higher IEP than for the controls. Electrophoretic mobility of the different samples of LDL was determined at pH 6.8 at different ionic strengths using NaCl or CaCl2. The effect of ionic strength (using NaCl) on the mobility was different for LDL isolated from different persons. Low concentrations of CaCl2 reverse the charge of lipoprotein from a negative to a positive one at pH 6.8. The drastic effect of Ca2+ on the net charge of LDL at pH 6.8, possibly due to the exposed phospholipid groups of LDL, shows that this ion may be important in the interaction between the lipoprotein and the mucopolysaccharides; such an interaction has been suggested by other workers as occurring in the arterial wall.

Urine steroid excretion in postmenopausal cancer of the breast. Response to corticotropin stimulation and dexamethasone suppression

Steroid hormone excretion patterns have been investigated in normal postmenopausal females and in subjects with mammary cancer with and without metastasis. Corticotropin (ACTH) stimulation and dexamethasone suppression tests were performed. Urinary 17‐ketosteroid (17‐KS), estrogens and pregnanediol were determined. Steroid hormone excretion varied widely between individuals under basal, ACTH‐stimulated and dexamethasone‐suppressed conditions. Subjects with mammary cancer differed from normal postmenopausal females in minor ways only. No clear cut excretion pattern was characteristic for a specific group. On ACTH stimulation the titer of estrogen and pregnanediol approached that of the premenopausal female. Dexamethasone failed to suppress estrogen excretion in a significant number of cancer patients. Dexamethasone did not suppress pregnanediol significantly in postmenopausal females or cancer patients. The level of excretion in the postmenopausal state is an individual characteristic and probably in unrelated to the carcinomatous process.

Regulation of lactate dehydrogenase gene expression by cAMP-dependent protein kinase subunits.

The studies described in this report suggest a rather complex, albeit incomplete, sequence of molecular events that we believe form part of the cascade of reactions through which a series of hormones, via cAMP, regulates the expression of specific gene products. The majority of our own studies relate to cAMP-mediated induction of LDH. Some, if not all, of the molecular steps discussed in this paper may ultimately be recognized as part of a universal mechanism by which cAMP controls gene expression in higher eukaryotes. The idea of a functional role for cAMP-dependent protein kinase subunits in cAMP-mediated gene control has already had experimental support, but our identification of the regulatory subunit RII as a topoisomerase now more firmly points to a complex function for the kinase in regulating gene function at the DNA level. We look forward to the elucidation of the function of those nuclear proteins that serve as substrate for the catalytic subunit of cAMP-dependent protein kinase. Further studies related to the molecular interaction of RII with chromosomal DNA should be a fruitful area for future research.

The Effect of Hormones on Hepatic Cholesterol Ester Synthesis in Vitro

Summary The effects of hormones on the in vitro biosynthesis of cholesterol palmitate, oleate, and linoleate by rat liver microsomes from cholesterol and fatty acids was studied. It appears that L-thyroxine and glucagon in general stimulate the synthesis of all three esters. Testosterone increases cholesterol palmitate and oleate formation. Its effect on cholesterol linoleate formation varies, being stimulatory at the highest concentration and inhibitory at the lowest concentration of hormone used. The action of 17 β-estradiol on cholesterol esterification also depended on the concentration of the hormone used. However, cholesterol oleate synthesis was markedly stimulated at all concentrations of 17β-estradiol.

Cyclic AMP-dependent and -independent protein phosphokinase activity in subcellular fractions of synchronously growing Leydig I-10 cells☆

Abstract The Leydig I-10 tumor cell line was synchronized by the double thymidine block method using 1.0 mM thymidine. Protein phosphokinase activity of subcellular fractions was determined at various times throughout the cell cycle. Microsomal cAMP-independent kinase activity increased in G2 and decreased during the S and G1 phases. Except for relatively small increases during the G1 and late S phases, microsomal cAMP-dependent kinase activity remained unchanged throughout most of the cycle. In the lysosomal-mitochondrial fraction, cAMP-dependent and cAMP-independent protein kinase activity increased during the S phase. Independent kinase activity peaked again during G1, while the dependent kinase became depressed. Phosphokinase activity increased in the nuclear fraction in late G2 and during mitosis, and was due to increases in both cAMP-independent and cAMP-dependent kinase activity. Cytosol cAMP-dependent kinase activity increased in G2 and during mitosis; cAMP-independent kinase activity showed some increased activity during late G2 and mitosis. These temporal variations in the subcellular kinase activities throughout the cell cycle may act to phosphorylate subcellular protein substrates in a cell cycle-specific fashion.

Ontogeny and multiplicity of cyclic AMP-dependent protein kinase in thymic lymphoid cells

Abstract Cyclic adenosine 3′:5′-monophosphate-dependent protein kinases were studied in thymus lymphoid cells and were found to be similar to their counterparts in other tissues with respect to substrate preference and concentration dependence. A previously not identified, restrictive subcellular compartmentalization of the protein kinase isozymes was found: Type I was predominantly present in the nucleus of adult and juvenile human and rat thymus cells, whereas the type II kinase was restricted to the cytosol fraction of unstimulated cells. Additionally, a decline in the specific activity of protein kinase was progressive with increasing age of the animal and distinct from the general observation that lymphoid cell numbers decrease with age. These findings may be correlated with age-dependent immunodeficiencies and perhaps have functional significance in the regulatory role of protein phosphorylation in lymphoid cell activation.

Hormonal effects on hepatic cholesterol biosynthesis and esterification.

Abstract The effects of castration and thyroidectomy on the hepatic concentration of free and esterified cholesterol and on the incorporation of acetate-1- 14 C and cholesterol- 3 H into cholesterol ester by intact liver and by a liver microsomal preparation have been studied in adult rats. After castration and thyroidectomy significantly lower concentrations of free cholesterol were found in the liver. The amount of liver cholesterol ester decreased after orchiectomy and increased after thyroidectomy. Ovariectomy had no effect on the ester concentration. To study cholesterol biosynthesis and esterification in vivo , endocrine ablated and control rats were injected with acetate-1- 14 C and cholesterol- 3 H and killed 1/2, 1, 2, 4 and 7 hr after injection. There was a decreased rate of incorporation of acetate-1- 14 C into liver cholesterol after ovariectomy, while orchiectomy resulted in increased rate of cholesterol synthesis. Esterification of cholesterol was decreased after thyroidectomy and ovariectomy, and increased after orchiectomy. In general, castration seemed to decrease the rate of turnover of hepatic free and esterified cholesterol. Throughout the in vivo experiments, the 14 C-labeled cholesterol ester fractions showed significantly higher specific activities than free cholesterol- 14 C. The preferential 14 C-labeling of the ester was probably due to an inhomogeneity and compartmentalization of hepatic cholesterol, and might indicate that only the most recently synthesized cholesterol- 14 C was used for esterification. In the in vitro experiments, hepatic microsomal enzyme preparations from thyroid-ectomized and castrated rats showed a higher activity of cholesterol esterification than the microsomal enzymes prepared from the normal control rats. Hormone replacement therapy abolished these differences in enzymatic activity.

17β-hydroxysteroid dehydrogenase activity in human thymus

Abstract Radioactive estrone, 17β-estradiol and androstenedione were incubated with human thymus homogenates. After isolation of the radioactive metabolites, estrone, 17β-estradiol, androstenedione and testosterone were characterized by isotope dilution, by chromatography on paper and silica gel, by the formation of derivatives, and by crystallization to constant specific activity. The findings show that human thymus homogenates are able to oxidize and reduce the 17-oxygen function in estrone, 17β-estradiol and androstenedione.

Effect of Protein Depletion on Response to Water, Sodium Chloride and Pituitrin

Summary 1. The time required to excrete 50% of an ingested water load was increased in 3 protein-depleted dogs. 2. The glomerular filtration rate was reduced in all protein-depleted dogs. 3. The urinary suppression produced by pituitrin was increased in the protein-depleted dogs.