Inhibiton of human placental 17 beta-hydroxysteroid dehydrogenase by steroids and nonsteroidal alcohols: aspects of inhibitor structure and binding specificity

Inhibition of human placental 17β-hydroxysteroid dehydrogenase by C₁₈ and C₁₉ steroids and nonsteroidal alcohols was assayed at pH 9.0 with 17β-estradiol 3-methyl ether and NAD⁺ as reactants. The nonstaroidal alcohols tested were poor inhibitors. Cyclopentanol and cyclohexanol had K_i values greater than 5 mm. Nonaromatic C₁₈ and C₁₉ steroids with oxygen functions at both positions 3 and 17 gave no detectable inhibition or had K_i, values greater than or equal to 160 μm. 3μ-Hydroxy-5,16-androstadiene, 5-androsten-3β-ol, 1,3,5(10)-estratrien-3-ol, and 1,3,5(10),16-estratetraen-3-ol, steroids lacking a C(17) oxygen function, had K_i values of 1.8, 6.0, 0.04, and 0.17 μm, respectively, demonstrating that both C₁₈ and C₁₉ steroids can bind at the steroid site. Binding specificity is narrowed and binding affinity for nonaromatic steroids weakened by oxygen functions at C(17) or both C(3) and C(17). The structural implications of the specificity data for steroid recognition and complex formation and in vivo control of enzyme activity are discussed.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

Inhibition of 17β-hydroxysteroid dehydrogenase (17β-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists

Various naturally occurring steroids, synthetic steroid derivatives and non-steroidal hormone agonists and antagonists were assayed as inhibitors of human placental 17β-HSD activities. Microsomal 17β-HSD was inhibited by C₁₈ -,C₁₉- and C₂₁-steroids. Soluble 17β-HSD was highly specific for C₁₈-steroids. In contrast to the soluble activity, the microsomal enzyme also had a strong affinity for ethinylestradiol (K_I=0.3 μM) and danazol (K_I=0.6 μM); anabolic steroids and norethisterone were weaker inhibitors. Of the non-steroids tested only diethylstilbestrol and o-demethyl CI-680 were inhibitors and they showed a greater affinity for soluble 17β-HSD.K_I-values for estradiol-17β, (0.8 μM), progesterone (27.0 μM) and 20α-dihydroprogesterone (1.5 μM) were comparable to reported tissue levels of these compounds, consistent with a possible competition in vivo among naturally occurring C₁₈-, C₁₉-, and C₂₁-steroids for the active site of microsomal 17β-HSD.     

Kinetic studies of the applicability of the common site concept to the 3β-hydroxysteroid dehydrogenase: 5-ene-3-ketosteroid isomerase activities of human placental microsomes

Pregnenolone (3β-hydroxy-5-pregnen-20-one) and DHA (3β-hydroxy-5-androsten-17-one), substrates for 3β-hy-droxysteroid dehydrogenase (3β-HSD), with K_M values of 15–40 nM, were ineffective inhibitors of 5-ene-3-ketosteroid isomerase (isomerase), with K_I values >40 μM in each case. Progesterone and androstenedione (4-androstene-3, 17-dione), 3β-HSD inhibitors with K_I values of 5.0 μM and 0.8 μM respectively, were also relatively ineffective inhibitors of isomerase, with K_I values of 30 μM and 16.5 μM respectively. Exposure of microsomes to hydrogen peroxide, which significantly increases the K_M for pregnenolone as a 3β-HSD substrate, had no effect on the K_M for the isomerase substrate 5-pregnene-3, 20-dione.It is concluded that the data do not support the common site concept with regard to the conversion of pregnenolone to progesterone by human placental microsomes.     

A simple method for detecting steroid aggregation and estimating solubility in aqueous solutions

Steroids are generally sparingly soluble in water. Thus, for in vitro studies of steroid metabolism or enzymology it is common practice to solubilize steroids by the addition of a small amount (2–10%, v/v) of an organic cosolvent. Methanol, ethanol, and 1,2-propanediol, singly or in combination, have been widely used (1). Effects of organic solvents on the kinetic parameters, K_m and V_max, of steroid-metabolizing enzymes with various substrates have been demonstrated (2,3), and the results are consistent with the conclusion that organic solvent influences on catalytic activity reflect, in part, effects on the aggregation state and solubility of steroid substrates.Light-scattering measurements have been applied extensively in studies of macromolecular structure (4) and micelle formation by a large variety of amphiphilic substances [reviewed in Ref. (5)]. Jones and Gordon (6) used a commercial instrument, designed specifically for light-scattering measurements, to characterize micelle formation in aqueous solutions by Δ5-3-ketosteroids containing various substituents at the 17β position. They showed that turbidity versus concentration plots were of the form seen in studies of micelle formation (5) and that steroids can exist in solution in monomeric or micellar forms, their aggregation state being a function of the polarity of the steroid solute and the composition of the solvent.To estimate solubility quantitatively ³H- or ¹⁴C-labeled steroids have been used in conjunction with centrifugation (3), dialysis (7), or filtration (8). These techniques allow for accurate estimates of solubility, but one may encounter problems due to nonspecific absorption on membranes or the unavailability of the labeled steroid of interest.We have observed that steroid aggregation and solubility can be estimated easily and with high sensitivity with a commercially available fluorometer. In this report the method is described and examples demonstrating the reproducibility and sensitivity of the technique are presented.     

The molecular weight and substrate specificity of 20 β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

The molecular weight of 20β-hydroxysteroid dehydrogenase was 111,000 when determined by agarose gel fitration and 106,000 by density gradient centrifugation. From gel electrophoresis in sodium dodecyl sulfate, after treatment with urea and 2-mercaptoethanol, the molecular weight was 27,000, consistent with the native molecule containing four subunits. After gel electrophoresis at pH 8.1, a single band was detected which stained for protein and activity with 5α-pregnan-20β-ol-3-one and 5α-androstan-3α,17β-diol. 20β-hydroxysteroid dehydrogenase was inactivated at pH 4.5 and the time course of inactivation was independent of the steroid used for activity measurements. Steroid substrates did not protect 20β-hydroxysteroid dehydrogenase against acid inactivation or affect enzyme fluorescence. It was concluded that the activity observed with the two substrates occurred at the same active center and that under the experimental conditions little steroid was bound to the enzyme in the the absence of coenzyme.     

Dedication to Dr. Li

When human placental microsomes were heated in boiling water or exposed to trypsin, 30 to 40% of the 5-ene,3-ketosteroid isomerase activity was stable. Aqueous suspensions of chloroform:methanol extracts of microsomes also catalyzed isomerization of 5-pregnene-3,20-dione, activity being associated with the polar lipid fraction. The trypsin- and heat-stable activities, as well as that of resuspended microsomal lipids, showed a dependence on buffer composition and concentration. Little activity was detected in water at pH 7.0. Relative activities in various buffers were Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) > Pipes (1,4-piperazinediethanesulfonic acid) > potassium phosphate > Mes(4-morpholineethanesulfonic acid). The data suggest that the occurrence of membrane lipid-dependent nonenzymatic catalysis could contribute to the isotope exchange with solvent observed in previous studies of the mechanism of isomerization catalyzed by placental microsomes. The ability of the membrane lipid phase to catalyze steroid isomerization under certain conditions and the fact that this activity is subject to modifications by exogenous agents may have more general implications for an understanding of possible effects of xenobiotics on steroid hormone formation and action in vivo.     

The influence of rotor speed on the sedimentation behavior in sucrose gradients of high molecular weight DNA's

Anomalous sedimentation behavior has been observed for high molecular weight duplex DNA's in sucrose gradients. The sedimentation rate of DNA's having molecular weights of 10⁸ or higher is influenced by high centrifugal fields. The change in the sucrose sedimentation coefficient due to this effect, S^RPM_suc-S⁰_suc, is equal to 1 × 10^?48M^3.65( The anomalous behavior is not influenced by DNA concentration at sufficiently low concentrations. Because of the smallness of the coefficient this effect has not been previously detected for DNA's the size of T2 or smaller at rotor speeds below 40000 RPM. For example, the relative sedimentation coefficient of T2 DNA at 65 000 RPM is only 9% less than at 10000 RPM. However, the sedimentation profile of heterogeneous high molecular weight [(100 – 350) × 10⁶] E. coli DNA is severely altered even at moderate rotor speeds (37000 RPM). Therefore, it seems advisable to use low rotor speeds when sedimenting high molecular weight DNA's.     

The explicit analysis of consecutive pseudo-first-order reactions: Application to kinetics of thiolysis of dithiasuccinoyl (Dts) amino acids

An explicit set of general methods for the experimental determination of the rates k₁ and k₂ of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity $\text{k}{}^{\mathrm{<mtext>env</mtext>}}\text{=}\text{k}{}_1\text{k}{}_2\text{(k}{}_1\text{+ k}{}_2\text{)}$ is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?k^envt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of $\text{κ =}\text{k}{}_2\text{k}{}_1$. The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory.     

Frequency and intensity of chick distress calls: Effects on maternal foodcalling in chickens

The food call of broody domestic hens was used to measure maternal response to four frequency components found in chick distress calls (2,3,4 and 5 kHz) and to variations in distress call intensity (0—86 dB). Foodcalling increased significantly with frequency of the pure-tone test pulse; response to a taped distress call occurred between 40 dB and 86 dB intensity with a maximum at 60–65 dB. The results suggest that the mother uses the higher frequency components in recognizing the distress call, but responds maximally within a specific intensity range. The selective advantage of such behaviour is discussed.     

Conversion of methionine to homocysteine thiolactone in liver

Since hemocysteinemia is associated with arteriosclerosis, the conversion of methione to homocysteine thiolactone was studied in guinea pig liver in vivo. 60 min after intraperitoneal injection of [¹⁴C]methione, [¹⁴C]homocystein thiolactone was found to constitute 9.1% ± 0.2 of the lipid bound ¹⁴C and 20% ± 1.0 of the acid soluble ¹⁴C. This conversion is the first step of a new pathway by which the sulfur of methionine is transferred to phosphoadenosine phosphosulfate.     

Analysis of double-helix motions with spin-labeled probes: binding geometry and the limit of torsional elasticity

The e.p.r.⁵ spectra of a family of spin-labeled probes non-covalently bound to DNA have been measured as functions of helix orientation, packing density and temperature. The spectra are interpreted in terms of the geometrical relations between the helix axis and the orbital containing the unpaired electron and in terms of the motions of the helix. Torsional and flexural motions can be distinguished.Spectra from well-ordered helices have been obtained using fully hydrated DNA fibers that are in thermodynamic equilibrium with unbound probe in dilute salt solution. The binding equilibria are similar to the equilibria in dilute DNA solution. The spatial relations between the spin label and the helix, inferred from the spectra, correspond closely to the structure expected on the basis of intercalation perpendicular to the helix axis and a sterically hindered amide bond between the spin label and the intercalating moiety of the probe. Viscometric measurements with one probe also indicate intercalation.Linear e.p.r. spectra of solutions, randomly condensed DNA, and fibers show substantial torsional motion but no detectable flexure on the linear e.p.r. time scale (> 300 ns). The correlation time of a propidium-based probe is much longer than that of aminoacridine intercalators. The probes with short correlation times are considered to be too weakly coupled to the adjacent base-pairs to be reliable indicators of DNA dynamics. For the propidium probe the correlation time, 30 nanoseconds, and its temperature dependence are compared with the properties expected according to four models: tight rotational coupling along the entire length of the helix; swivels at fixed intervals; a two-state exchange; and elastic rotational coupling between adjacent nucleotide pairs. In terms of the fourth model, the results suggest that each nucleotide pair undergoes random oscillation with an r.m.s. amplitude of not more than 4 ° to 5 ° at room temperature. That value agrees with estimates made in other ways.     

Iridodial,and a new alkanone, 4-methylhexan-3-one,in the defensive secretion of the beetle, Staphylinus olens

The monoterpene iridodial and the ketone, 4-methylhexan-3-one, have been identified as the major components of the defensive secretion from Staphylinus olens.     

Symbolic logic analysis of cause of dealth in humans: Application to 108 patients after coronary artery bypass graft surgery

Over the past two decades there has been increasing interest in the development of an objective, or formalized “medical logic”, and many authors have employed classical symbolic logic as a part of their approach. On the other hand, it has become clear that certain patterns of reasoning which are commonplace in evaluating patients clinicopathologically are awkward to handle in classical symbolic logic. The present paper proposes an extension of classical symbolic logic which addresses three problems in medical reasoning: (i) the problem of provisional diagnosis, (ii) the problem of inaccessible data, and (iii) the problem of the adequate discharge summary. It is proved mathematically that with a suitably constructed logic, the system “complains” until all questions involving threats to the patient's health are either answered or shown to be unanswerable because of inaccessibility of data. To illustrate this method, the cause of death was studied in 108 patients who had been autopsied at The Johns Hopkins Hospital after coronary artery bypass surgery. The analysis disclosed that 46% of patients suffered a fatal complication which could be attributed to events in the perioperative period; in 15% of patients the cause of death was unexplained by the analysis. Computerized symbolic logic analysis is a useful supplement to intuitive reasoning in assigning cause of death to patients with complex medical histories.     

Production of prostaglandins by cells in vitro: radioimmunoassay measurement of the conversion of arachidonic acid to PGE2 and PGF2 alpha

A specific radioimmunoassay has been applied to the measurement of the conversion of arachidonic acid to PGE₂ and PGF_2α. PGE₂ and PGF_2α biosynthesis was linearly related to the amount of arachidonic acid added and was significantly inhibited by indomethacin in concentrations as low as 10^?10 M. Sonicated Hela, L, and HEp-2 cells synthesized 244.0, 42.3, and 22.6 ng PGE₂ per mg of protein, but made substantially less PGF_2α.     

Myocardial infarction in the guinea pig: cellular electrophysiology

The cellular electrophysiology of left ventricular preparations from guinea pig hearts was studied 1 hour, 24 hours, and 4-6 weeks after myocardial infarction produced by 6-8 single ties of the distal left coronary artery system or after sham operation. Microelectrode recordings were used to monitor cells from the endocardial surface of each preparation in tissue bath. All coronary ligated preparations displayed accelerated spontaneous activity compared to normal and sham operated preparations. Single and multiple premature ventricular depolarizations occurred frequently in coronary ligated and rarely in normal and sham operated preparations. Premature stimuli delivered to areas overlying and bordering the area of infarction, induced short bursts of self-terminating rapid repetitive ventricular activity in 4 of 8 (50%) acute (1-hour), 5 of 9 (55%) subacute (24-hour), and 14 of 20 (70%) healed (4-6-week) infarcted preparations. Such activity could not be induced in normal and sham operated preparations. The preparations with healed infarction were unique in that they demonstrated runs of self-terminating repetitive ventricular activity which occurred spontaneously or was inducible with premature stimulation. Recordings from multiple sites in acute, subacute, and healed preparations revealed a variety of transmembrane action potential abnormalities (i.e., reduced action potential amplitude and resting potential, decreased and increased action potential duration, and depressed maximum rates of phase 0 depolarization) in cells overlying and bordering areas of infarction. Only Purkinje fiber action potentials were recorded over the healed infarcts. These data demonstrate that a spectrum of electrophysiological alterations occur in response to ischemic injury and persist after healing of the injury in this new model of myocardial infarction utilizing the guinea pig.     

Effects of estrogen deprivation on brain estrogen and progestin receptor levels and the activation of female sexual behavior

Ovariectomy (OVX) in rats is followed by a decline in behavioral sensitivity to combined estrogen and progesterone therapy. The purpose of this study was to further characterize this behavioral change, and to explore its biochemical basis in terms of estrogen and progesterone receptor concentrations in the brain. Sexually inexperienced female rats were used 5 (short-term) or 35 (long-term) days after OVX. Short- and long-term OVX animals were injected with estradiol-17β (E₂; 36 μg/kg body wt, iv) then subjected to one of the following three treatment schedules. (1) Animals were treated with progesterone (1 mg, sc in oil) 20–21 hr after E₂ injection, then tested at 24 hr for female sexual behavior. (2) One or twelve hours after the E₂, cell nuclear estrogen receptors (ER_n) were measured in the pituitary (PIT) and pooled preoptic area and mediobasal hypothalamus (POA-MBH). (3) Twenty-four hours after E₂, progestin receptor (PR_c) concentrations were measured in cytoplasmic fractions prepared from PIT and POA-MBH. Long-term OVX animals showed a reduced capacity to exhibit proceptive and receptive sexual behavior, and a lower PR_c level in the PIT and POA-MBH 24 hr after E₂ injection than animals that had been OVX for only 5 days. However, no differences were observed between long- and short-term OVX rats with respect to ER_n concentrations in PIT and POA-MBH cell nuclei 1 or 12 hr after E₂. Thus, it appears that the decline in behavioral responsiveness to E₂ which occurs after ovariectomy cannot be attributed to a decrease in the ability of E₂ to translocate estrogen receptors into POA-MBH cell nuclei, but is more probably associated with a change in the biochemical processes subsequent to ER_n binding. One of these processes may well be the induction of cytoplasmic progestin receptors.     

Chromosomes of mouse oocytes in maturation: differential trypsin sensitivity and amino acid incorporation.

In the period of maturation in vivo, the chromosomes of mouse oocytes display a spectrum of unique configurations that is postulated to be related to a sequence of turnover of chromosomal proteins. Evidence on behalf of that hypothesis is provided by the following cytologic observations: The chromosomes of the diakinesis-metaphase I complement are resistant to disruption by mild treatment with trypsin. Following metaphase I, the chromosomes become exceedingly compact and display correlated increased resistance to trypsin. At telophase I, when the complements of the secondary oocyte and the first polar body have each coalesced into a “chromatin mass,” the chromosomes are greatly sensitive to trypsin. Following separation from the mass, the definitive oocyte chromosomes decompact into a “relaxed coil” conformation and display moderate trypsin sensitivity comparable to that of mitotic metaphase chromosomes. Autoradiography of [³H]-arginine and [³H]tryptophan incorporation show that while both amino acids are incorporated into the ooplasm, arginine, but not tryptophan, is incorporated into the chromosomal material. Analysis of the data indicates that incorporation takes place as two separate events, one in late dictyotene and the other post-telophase I and that the arginine-containing proteins incorporated into the dictyate chromosomes are transient and are not retained on the metaphase II chromosomes.