7-Hydroxydehydroepiandrosterone--a natural antiglucocorticoid and a candidate for steroid replacement therapy?

7-Hydroxylated metabolites of dehydroepiandrosterone (DHEA) are believed to be responsible for at least some immunomodulatory and antiglucocorticoid effects of DHEA and hence are considered candidates for hormone replacement therapy. Our experiments in vitro brought the evidence that 3beta, 7beta-dihydroxy-5-androsten-3-one (7beta-OH-DHEA), but not DHEA and its 7alpha-hydroxyisomer, could counteract the immunosuppressive effect of dexamethasone on the formation of plaques in culture of murine spleen lymphocytes. In another experiment, DHEA and after a 3-weeks pause 3beta-hydroxy-5-androstene-7,17-dione (7-oxo-DHEA) were applied transdermally to 6 male volunteers on 5 consecutive days. Blood levels of DHEA, its 7-hydroxylated metabolites, and in the first case also dehydroepiandrosterone sulphate (DHEAS), were measured before, during and one day after the end of treatment. Application of DHEA increased significantly not only DHEA and DHEAS, but also its both 7-hydroxyisomers. Application of 7-oxo-DHEA also led to a significant increase of both 7-hydroxyisomers of DHEA, with 7beta-OH-DHEA being the preferred metabolite the concentration of which was increased more than three times.     

Effects of night shift on the cognitive load of physicians and urinary steroid hormone profiles – a randomized crossover trial

Mental and physical stress is common in physicians during night shifts. Neurocognitive effects of sleep deprivation as well as alterations in hormonal and metabolic parameters have previously been described. The aim of this crossover study was to evaluate the effects of night-shift work with partial sleep deprivation on steroid hormone excretion and possible associations with mood, sleep characteristics and cognitive functions in physicians.

In total, 34 physicians (mean age 42 ± 8.5 years, 76.5% male) from different departments of the General Hospital of Vienna, Austria, were randomly assigned to two conditions: a regular day shift (8 h on duty, condition 1) and a continuous day-night shift (24 h on duty, condition 2). In both conditions, physicians collected a 24 h urine sample for steroid hormone concentration analysis and further completed psychological tests, including the sleep questionnaire (SF-A), the questionnaire for mental state (MDBF) and the computer-assisted visual memory test (FVW) before and at the end of their shifts, respectively.

Although mean sleep deprivation during night shift was relatively small (~1.5 h) the impairment in participants’ mental state was high in all three dimensions (mood, vigilance and agitation, p ≤ 0.001). Sleep quality (SQ), feeling of being recovered after sleep and mental balance decreased (p ≤ 0.001), whereas mental exhaustion increased (p < 0.05). Moreover, we could show a nearly linear relationship between most of these self-rating items. Testing visual memory participants made significantly more mistakes after night shift (p = 0.011), however, mostly in incorrectly identified items and not in correctly identified ones (FVW). SQ and false identified items were negatively correlated, whereas SQ and time of reaction were positively associated. It is assumed that after night shift, a tendency exists to make faster wrong decisions. SQ did not influence correctly identified items in FVW. In contrast to previous investigations, we found that only excretion rates for pregnanetriol and androsterone/etiocholanolone ratios (p < 0.05, respectively) were slightly reduced in 24-h urine samples after night shift. A considerable stimulation of the adrenocortical axis could not be affirmed. In general, dehydroepiandrosteron (DHEA) was negatively associated with the sense of recreation after sleep and with the time of reaction and positively correlated with correctly identified items in the FVW test.

These results, on the one hand, are in line with previous findings indicating that stress and sleep deprivation suppress gonadal steroids, but, on the other hand, do not imply significant adrenocortical-axis stimulation (e.g. an increase of cortisol) during the day-night shift.     

Isolation and characterization of 17β-hydroxy steroid dehydrogenase from human erythrocytes

1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.     

Structure and Properties of a Complex of α-Synuclein and a Single-Domain Camelid Antibody

The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.     

Activity of steroid 4 and derivatives 4a–4f as inhibitors of the enzyme 5α-reductase 1

It is known that the growth of prostate metastatic bone tumor depends on androgens, and tumor formation can start from migratory malignant cells produced in that organ. These cells exhibit grater type 1 5α-reductase (5α-R1) activity than type 2 5α-reductase. Noteworthy, both isozymes convert testosterone (T) to the more active androgen dihydrotestosterone (DHT) in the target tissues.Thus, in order to potentially improve the prognosis of this disease, in this work, seven derivatives of 17-(1H-benzimidazol-1-yl)-16-formillandrosta-5,16-dien-3β-yl benzoate (4a–f) and 17-(1H-benzimidazol-1-yl)-3-hydroxy-16-formylandrost-5,16-diene (4) were synthesized, characterized and identified as inhibitors of type 1 5α-reductase (5αR1). These derivatives having the advantage of improved plasma half-life.The inhibitory activity of the compounds towards 5α-R1 isoenzyme was determined by conversion of T into DHT in the presence or absence of compounds 4, 4a–f. Further, in vivo experiments were also carried out, treating gonadectomized hamsters with T and/or 4, 4a–f and evaluating their effect on the diameter of hamster flank organs and on the weight of the prostatic and seminal vesicles. Results indicated that compounds 4, 4b, 4c, served as in vitro inhibitors of the enzyme 5α-R1 and pharmacological experiments showed that 4 and derivatives 4a–f decreased the diameter of the flank glands, the weight of the prostate and seminal vesicles of treated hamsters without any appreciable toxicity during observation. Noteworthy the fact that compound 4 is the product, in all cases, of the hydrolysis of the series of esters 4a–f, thus they can serve as precursors (prodrugs) of the active form 4.     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

Revealing a steroid receptor ligand as a unique PPARγ agonist

Peroxisome proliferator-activated receptor gamma (PPARγ) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPARγ agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPARγ target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPARγ ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPARγ ligands in the treatment of insulin resistance.     

Crystallization and identification of a binary complex of a elastase-I and a Carboxypeptidase Aγ from porcine pancreas

A binary complex of elastase I and Carboxypeptidase Aγ has been isolated and crystallized from activated extracts of porcine pancreas. The purification procedure included ammonium sulfate fractionation and autolysis treatment followed by crystallization. The two components in the complex have been separated by DEAE-Sephadex A-50 column chromatography to homogeneity and their identification was demonstrated by NH₂-terminal sequence analysis. An analysis of the reconstitution crystal from the mixtures of both components showed that the complex consisted of a 1:1 molar ratio with elastase I and Carboxypeptidase Aγ, forming a binary complex in crystalline state.     

Pregna-D′-pentaranes,progestins and antiprogestins: I. Separation of biological functions of steroid hormones

The synthesis, modification, structure, and biological activity in vivo of the 16,17- cycloalkanoprogesterone (pregna-D-pentarane) analogues of progesterone are described. A possibility of separation of their biological functions has been demonstrated. A systematic synthesis of a set of uniform compounds that differ in a limited number of alterable parameters was developed. It resulted in an instrument useful for the investigation of pathways and mechanisms by which the steroid hormones fulfill their biological functions and for the probable discovery of new functions masked by the wide effects of native compounds.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 115–129.Original Russian Text Copyright © 2005 by Kamernitzky, Levina.THE CYCLE OF WORKS OF THE AUTHORS DESCRIBED IN THE REVIEW HAS WON THE SHEMYAKIN PRIZE IN 2004     

BF₃·Et₂O-induced stereoselective aldol reaction with benzaldehyde, and steroid sapogenins and its application to a convenient synthesis of dinorcholanic lactones

Treatment of steroid sapogenins with benzaldehyde and BF(3)·Et(2)O cleanly produces E-23(23')-benzylidenspirostanes in good yields in a reaction pathway which consists on an aldol reaction followed by a dehydration step. The obtained E-23(23')-benzylidenspirostanes can be easily converted to dinorcholanic lactones by treatment with CrO(3) in acetic acid. The synthetic sequence to dinorcholanic lactones is compatible with the presence of double bonds and carbonyl groups in the steroid framework.     

Specificity in steroid histochemistry,with special reference to the use of steroid solvents. distribution of 11 β-hydroxysteroiddehydrogenase in kidney and thymus from the mouse

Summary Several factors influencing the steroiddehydrogenase histochemistry were investigated: diffusion of enzyme; inactivation of enzyme; effects of the steroid solvents commonly used; the validity in localization of the enzyme activity; nothing dehydrogenase reaction. 1. The importance in controlling the diffusion of each enzyme system to be studied is emphasized. Provided that the presence of SH-groups in the active centre of the dehydrogenase can be proved, a control experiment using a double-section incubation method should be carried out. 2. A comparison between the use of unfixed and briefly prefixed sections is recommended in order to avoid a possible distortion of the tissue during the incubation. The influence of prefixation on diffusion of enzymes or reaction products as well as on inactivation of enzymes must be studied. 3. The steroid solvents—especially dimethyl formamide caused a morphological distortion, and an inactivation and/or extraction of reaction products (the red monoformazan) in fresh frozen sections, these solvents should therefore be handled with caution. A special mixture of dimethyl formamide and propylene glycol is recommended. 4. The steroid should be completely soluble in the incubation medium in order to secure zero order kinetics. 5. Avoidance of sulphydryl nothing dehydrogenase reaction, since the reaction predominantly manifests itself as a red formazan obscuring sites with low dehydrogenase activity. 6. The localization of the NAD(P)H oxidase systems must be controlled, in order to ensure that they should not be a limiting factor in the detection of the dehydrogenase activity. Secondly, this investigation may act as a control on diffusion of dehydrogenase and/or reduced coenzyme. 7. That the investigation of the incubation time needed for initial visual reaction allows a certain quantitative estimation of the concentration of enzyme localized at different sites in the same section. The investigation should also include the red formazan, since it has recently been proved to be an intermediary step in the enzymic reduction of Nitro BT, and as such may reflect sites with low enzyme concentration.Further, some of the functional aspects of the activity of 11-hydroxysteroiddehydrogenaseNAD(P)H oxidase systems in the thymus were discussed, and lastly the localization of these systems in the kidney was revised.This work was supported by a grant from Statens almindelige Videnskabsfond, Copenhagen.     

8-Phenylthio-adenines stimulate the expression of steroid hydroxylases, Cav3.2 Ca²⁺ channels, and cortisol synthesis by a cAMP-independent mechanism

The regulation of cortisol synthesis and the expression of genes coding for steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10-50 μM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2'-OMe-cAMP, but not 8HPT-2'-OMe-cAMP or 8MeOPT-2'-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 steroid hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10-200 μM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca(2+) antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Ca(v)3.2 Ca(2+) current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Ca(v)3.2 Ca(2+) channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.     

The use of deoxyribonucleic acid–cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid–receptor complexes

1. Two characteristic properties of the specific high-affinity steroid-binding proteins or receptors, their ability to bind to DNA-cellulose and their relatively acidic isoelectric point, have been exploited as a means of purification. These two fundamental properties distinguish the receptors from the steroid-binding proteins in serum and the non-specific low-affinity steroid-binding proteins in hormone-responsive cells. 2. A significant degree of purification of both cytoplasmic and nuclear steroid-receptor complexes can be achieved with practical facility by these procedures. The purity of the receptor complexes is sufficient to enable studies on their possible control of metabolic processes to be investigated in the future. 3. After extensive purification the physicochemical properties of the cytoplasmic androgen-receptor complex, such as sedimentation coefficient, were unchanged. Further, the purified complex fully retained at least one of its fundamental physiological properties, namely the ability to transfer 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) into chromatin in vitro. 4. The methods may also be employed for studying the changes in the structure and properties of the receptor complexes that are an essential prerequisite for the transfer of cytoplasmic receptor complexes into nuclear chromatin. The temperature-dependence of the binding of androgen-receptor complexes into chromatin is essentially due to a major change in cytoplasmic receptor complex before its attachment to nuclear chromatin. 5. The resolution of these analytical procedures was sufficient to enable a critical comparison of the receptor proteins from different male accessory glands to be undertaken. From these studies, no substantial evidence in support of the tissue specificity of androgen receptors could be established; rather the receptors from different androgen-dependent glands were remarkably similar in physicochemical properties. 6. Although the methods were initially developed for the partial purification of androgen-receptor complexes, they are equally suitable for the prompt and extensive purification of oestrogen-receptor and progesterone-receptor complexes.     

Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs--involvement of steroid hormones

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E₂) and progesterone (P₄) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E₂ on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E₂ and P₄ level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.     

A diastereoselective synthesis of 7α-nitromethyl steroid derivative and its use for an efficient synthesis of eplerenone

A novel and efficient method of stereoselectively introducing α-nitromethyl group to C-7 position of 11α-hydroxyl canrenone 4 was described. In addition, this method was successfully applied in a total synthesis of Eplerenone 8. The route was characteristic of simple operation, moderate reaction conditions with 5 steps and 55% total yield, at the same time, without any expensive or toxic reagent in use.     

Synthesis and moisture absorption and retention activities of a carboxymethyl and a quaternary ammonium derivative of α,α-trehalose

Carboxymethyl α,α-trehalose (CMT) and a quaternary ammonium derivative of α,α-trehalose (QT) were successfully prepared, and their moisture absorption and retention activities were assessed. Results showed that both CMT and QT had better moisture absorption abilities at 43% and 81% relative humidity (RH) than α,α-trehalose. In addition, the two α,α-trehalose derivatives had better moisture retention abilities than α,α-trehalose under three humidity conditions: 81% RH, 43% RH, and under dry conditions. Therefore, carboxymethylation and quaternarization could improve the moisture absorption and retention abilities of α,α-trehalose. CMT and QT showed better moisture absorption ability and moisture retention ability than that of hyaluronan (HA), and could potentially find a use as moisture retention ingredient, for example, in cosmetics.     

Design and synthesis of a vialinin A analog with a potent inhibitory activity of TNF-α production and its transformation into a couple of bioprobes

Vialinin A (1) is an extremely potent inhibitor against tumor necrosis factor (TNF)-α production in rat basophilic leukemia (RBL-2H3) cells. This Letter describes the design and synthesis of its advanced analog, 5',6'-dimethyl-1,1':4'1″-terphenyl-2',3',4,4″-tetraol (2) with a comparable inhibitory activity (IC(50)=0.02 nM) to that of 1. The synthesis involved double Suzuki-Miyaura coupling as a key step, and required only five steps from commercially available 3,4-dimethylphenol. For identification of the target molecule, fluorescent and biotinylated derivatives of 2 were prepared through a 'click' coupling process.