Mechanism of the Dual Activities of Human CYP17A1 and Binding to Anti-Prostate Cancer Drug Abiraterone Revealed by a Novel V366M Mutation Causing 17,20 Lyase Deficiency.

The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA) was impaired, explaining the patient’s steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD) simulations showed that V366M creates a “one-way valve” and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therapy.

Androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway: Insights into enzyme activities and steroid fluxes in healthy infants during the first year of life from the urinary steroid metabolome.

The steroid profile changes dramatically from prenatal to postnatal life. Recently, a novel backdoor pathway for androgen biosynthesis has been discovered. However, its role remains elusive. Therefore, we investigated androgen production from birth to one year of life with a focus on minipuberty and on production of androgens through the backdoor pathway. Additionally, we assessed the development of the specific steroid enzyme activities in early life. To do so, we collected urine specimens from diapers in 43 healthy newborns (22 females) at 13 time points from birth to one year of age in an ambulatory setting, and performed in house GC-MS steroid profiling for 67 steroid metabolites. Data were analyzed for androgen production through the classic and backdoor pathway and calculations of diagnostic ratios for steroid enzyme activities were performed. Analysis revealed that during minipuberty androgen production is much higher in boys than in girls (e.g. androsterone (An)), originates largely from the testis (Anboys-Angirls), and uses predominantly the alternative backdoor pathway (An/Et; Δ5<Δ4 lyase activity). Modelling of steroid enzyme activities showed age-related effects for 21-, 11-, 17-hydroxylase and P450 oxidoreductase activities as well as 3ß-hydroxysteroid dehydrogenase, 11ß-hydroxylase type 1/2 and 5α-reductase activities. Sex-related characteristics were found for 21-hydroxylase and 5α-reductase activities. Overall, our study shows that androgen biosynthesis during minipuberty favors the backdoor pathway over the classic pathway. Calculations of specific diagnostic ratios for enzyme activities seem to allow the diagnosis of specific steroid disorders from the urinary steroid metabolome.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children: Tracing the complexity of human postnatal steroidogenesis.

CONTEXT: Complex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by (a) the metabolites of the fetal-placental unit at birth, (b) the fetal adrenal androgens until its involution 3-6 months postnatally, and (c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. OBJECTIVE: The aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. METHODS: We conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. RESULTS: 61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. CONCLUSION: The urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.

Detection and functional portrayal of a novel class of dihydrotestosterone derived selective progesterone receptor modulators (SPRM).

In early pregnancy, abortion can be induced by blocking the actions of progesterone receptors (PR). However, the PR antagonist, mifepristone (RU38486), is rather unselective in clinical use because it also cross-reacts with other nuclear receptors. Since the ligand-binding domain of human progesterone receptor (hPR) and androgen receptor (hAR) share 54% identity, we hypothesized that derivatives of dihydrotestosterone (DHT), the cognate ligand for hAR, might also regulate the hPR. Compounds designed and synthesized in our laboratory were investigated for their affinities for hPRB, hAR, glucocorticoid receptor (hGRα) and mineralocorticoid receptor (hMR), using whole cell receptor competitive binding assays. Agonistic and antagonistic activities were characterized by reporter assays. Nuclear translocation was monitored using cherry-hPRB and GFP-hAR chimeric receptors. Cytostatic properties and apoptosis were tested on breast cancer cells (MCF7, T-47D). One compound presented a favorable profile with an apparent neutral hPRB antagonistic function, a selective cherry-hPRB nuclear translocation and a cytostatic effect. 3D models of human PR and AR with this ligand were constructed to investigate the molecular basis of selectivity. Our data suggest that these novel DHT-derivatives provide suitable templates for the development of new selective steroidal hPR antagonists.

18F-RB390: innovative ligand for imaging the T877A androgen receptor mutant in prostate cancer via positron emission tomography (PET).

BACKGROUND: Detecting prostate cancer before spreading or predicting a favorable therapy are challenging issues for impacting patient's survival. Presently, 2-[(18) F]-fluoro-2-deoxy-D-glucose ((18) F-FDG) and/or (18) F-fluorocholine ((18) F-FCH) are the generally used PET-tracers in oncology yet do not emphasize the T877A androgen receptor (AR) mutation being exclusively present in cancerous tissue and escaping androgen deprivation treatment. METHODS: We designed and synthesized fluorinated 5α-dihydrotestosterone (DHT) derivatives to target T877A-AR. We performed binding assays to select suitable candidates using COS-7 cells transfected with wild-type or T877A AR (WT-AR, T877A-AR) expressing plasmids and investigated cellular uptake of candidate (18) F-RB390. Stability, biodistribution analyses and PET-Imaging were assessed by injecting (18) F-RB390 (10MBq), with and without co-injection of an excess of unlabeled DHT in C4-2 and PC-3 tumor bearing male SCID mice (n = 12). RESULTS: RB390 presented a higher relative binding affinity (RBA) (28.1%, IC50 = 32 nM) for T877A-AR than for WT-AR (1.7%, IC50 = 357 nM) related to DHT (RBA = 100%). A small fraction of (18) F-RB390 was metabolized when incubated with murine liver homogenate or human blood for 3 hr. The metabolite of RB390, 3-hydroxysteroid RB448, presented similar binding characteristics as RB390. (18) F-RB390 but not (18) F-FDG or (18) F-FCH accumulated 2.5× more in COS-7 cells transfected with pSG5AR-T877A than with control plasmid. Accumulation was reduced with an excess of DHT. PET/CT imaging and biodistribution studies revealed a significantly higher uptake of (18) F-RB390 in T877A mutation positive xenografts compared to PC-3 control tumors. This effect was blunted with DHT. CONCLUSION: Given the differential binding capacity and the favorable radioactivity pattern, (18) F-RB390 represents the portrayal of the first imaging ligand with predictive potential for mutant T877A-AR in prostate cancer for guiding therapy. Prostate 75:348-359, 2015. © 2014 Wiley Periodicals, Inc.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11ß-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11ß-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.

Regulation of 11ß-hydroxysteroid dehydrogenase type 2 by microRNA.

The enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) is selectively expressed in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor. A diminished activity causes salt-sensitive hypertension. The mechanism of the variable and distinct 11ß-hydroxysteroid dehydrogenase type 2 gene (HSD11B2) expression in the cortical collecting duct is poorly understood. Here, we analyzed for the first time whether the 11ß-HSD2 expression is modulated by microRNAs (miRNAs). In silico analysis revealed 53 and 27 miRNAs with potential binding sites on human or rat HSD11B2 3'-untranslated region. A reporter assay demonstrated 3'-untranslated region-dependent regulation of human and rodent HSD11B2. miRNAs were profiled from cortical collecting ducts and proximal convoluted tubules. Bioinformatic analyses showed a distinct clustering for cortical collecting ducts and proximal convoluted tubules with 53 of 375 miRNAs, where 13 were predicted to bind to the rat HSD11B2 3'-untranslated region. To gain insight into potentially relevant miRNAs in vivo, we investigated 2 models with differential 11ß-HSD2 activity linked with salt-sensitive hypertension. (1) Comparing Sprague-Dawley with low and Wistar rats with high 11ß-HSD2 activity revealed rno-miR-20a-5p, rno-miR-19b-3p, and rno-miR-190a-5p to be differentially expressed. (2) Uninephrectomy lowered 11ß-HSD2 activity in the residual kidney with differentially expressed rno-miR-19b-3p, rno-miR-29b-3p, and rno-miR-26-5p. In conclusion, miRNA-dependent mechanisms seem to modulate 11ß-HSD2 dosage in health and disease states.

Major cardiac surgery induces an increase in sex steroids in prepubertal children.

While the neuroprotective benefits of estrogen and progesterone in critical illness are well established, the data regarding the effects of androgens are conflicting. Surgical repair of congenital heart disease is associated with significant morbidity and mortality, but there are scant data regarding the postoperative metabolism of sex steroids in this setting. The objective of this prospective observational study was to compare the postoperative sex steroid patterns in pediatric patients undergoing major cardiac surgery (MCS) versus those undergoing less intensive non-cardiac surgery. Urinary excretion rates of estrogen, progesterone, and androgen metabolites (µg/mmol creatinine/m(2) body surface area) were determined in 24-h urine samples before and after surgery using gas chromatography-mass spectrometry in 29 children undergoing scheduled MCS and in 17 control children undergoing conventional non-cardiac surgery. Eight of the MCS patients had Down's syndrome. There were no significant differences in age, weight, or sex between the groups. Seven patients from the MCS group showed multi-organ dysfunction after surgery. Before surgery, the median concentrations of 17ß-estradiol, pregnanediol, 5α-dihydrotestosterone (DHT), and dehydroepiandrosterone (DHEA) were (control/MCS) 0.1/0.1 (NS), 12.4/11.3 (NS), 4.7/4.4 (NS), and 2.9/1.1 (p=0.02). Postoperatively, the median delta 17ß-estradiol, delta pregnanediol, delta DHT, and delta DHEA were (control/MCS) 0.2/6.4 (p=0.0002), -3.2/23.4 (p=0.013), -0.6/3.7 (p=0.0004), and 0.5/4.2 (p=0.004). Postoperative changes did not differ according to sex. We conclude that MCS, but not less intensive non-cardiac surgery, induced a distinct postoperative increase in sex steroid levels. These findings suggest that sex steroids have a role in postoperative metabolism following MCS in prepubertal children.

Evidence for glucocorticoid-mediated hypertension after uninephrectomy.

Recently, evidence was presented that uninephrectomy induces salt-sensitive hypertension in rats. The increase in blood pressure was abrogated by a mineralocorticoid receptor antagonist but not by an aldosterone synthase inhibitor. Here, we hypothesize that mineralocorticoid receptor activation occurred by an 11beta-hydroxy-glucocorticosteroid, as a consequence of dysregulated 11beta-hydroxysteroid dehydrogenase enzymes. Therefore, 3-week-old Sprague-Dawley rats were either uninephrectomized or sham operated and given a normal (0.4%) or high (8%)-salt diet. At week 8, a telemetric device was implanted, and during week 13 blood pressure continuously measured and urine was collected. The animals were sacrificed thereafter and liver and kidney were harvested. Steroid metabolites were analyzed by GC-MS and mRNA assessed by PCR. Uninephrectomy caused a distinct salt-sensitive hypertension. The increase in blood pressure correlated significantly with a decline in the apparent activity of 11beta-hydroxysteroid dehydrogenase 2 and an increase of 11beta-hydroxysteroid dehydrogenase 1, when urinary corticosterone metabolites were considered. These results were mirrored by the corresponding metabolite ratios assessed in renal and liver tissue. Changes in enzyme activities were in part explained by changes in the mRNA content. In conclusion, mineralocorticoid receptor-dependent salt sensitivity after UNX in rats appears to be mediated by glucocorticoids.

Vascular endothelial growth factor-A and aldosterone: relevance to normal pregnancy and preeclampsia.

Aldosterone levels are markedly elevated during normal pregnancy but fall even though volume contracts when preeclampsia occurs. The level of aldosterone in either condition cannot be explained solely by the activity of the renin-angiotensin II system. In normal gestation, vascular endothelial growth factor (VEGF) is thought to maintain vascular health, but its role in adrenal hormone production is unknown. We hypothesized that the role of VEGF in the adrenal gland is to maintain vascular health and regulate aldosterone production. Here, we demonstrate that supernatant of endothelial cells grown in the presence of VEGF enhanced aldosterone synthase activity in human adrenocortical cells. VEGF either alone or combined with angiotensin II increased aldosterone production in adrenal cells. These data suggest that endothelial cell-dependent and independent activation of aldosterone is regulated by VEGF. In contrast to angiotensin II, VEGF did not upregulate the steroidogenic acute regulatory protein. Consistent with this observation, angiotensin II stimulated both aldosterone and cortisol synthesis from progesterone, whereas VEGF stimulated selectively aldosterone production. In rats, overexpression of soluble fms-like tyrosine kinase-1, an endogenous VEGF inhibitor, led to adrenocortical capillary rarefaction and fall in aldosterone concentrations that correlated inversely with soluble fms-like tyrosine kinase-1 levels. These findings may explain why aldosterone increases so markedly during normal gestation and why preeclampsia, a condition characterized by high soluble fms-like tyrosine kinase-1, is associated with inappropriately low aldosterone levels in spite of relatively lower plasma volumes.

High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11ß-hydroxysteroid dehydrogenase type 2.

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11ß-hydroxy-glucocorticoids is modulated by the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). Here we present evidence for intestinal segment specific 11ß-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11ß-HSD2, MR and ENaC expression. The 11ß-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11ß-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-ß and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

A novel steroidal antiandrogen targeting wild type and mutant androgen receptors.

Prostate cancer (PCa) progression is enhanced by androgen and treatment with antiandrogens represents an alternative to castration. While patients initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years by expressing androgen receptor (AR) mutants. Such mutations, indeed, promote unfavorable agonistic behavior from classical antagonists. Here, we have synthesized and screened 37 novel compounds derived from dihydrotestosterone (DHT), cyanolutamide and hydroxyflutamide. These derivatives were tested for their potential antagonistic activity using a luciferase reporter gene assay and binding properties were determined for wild type (WT) and mutant ARs (T877A, W741C, W741L, H874Y). In the absence and presence of antiandrogens, androgen dependent cellular proliferation and prostate specific antigen (PSA) expression were assayed in the prostate cancer cell line LNCaP by crystal violet, real time PCR and by Western blots. Also, cellular proliferation and PSA expression were assayed in 22Rv1. A novel compound RB346, derived from DHT, was found to be an antagonist for all tested AR forms, preventing DHT induced proliferation and PSA expression in LNCaP and 22Rv1 cells. RB346 displayed no agonistic activity, in contrast to the non-steroidal antiandrogen bicalutamide (Casodex) with unfavorable agonistic activity for W741L-AR. Additionally, RB346 has a slightly higher binding affinity for WT-AR, T877A-AR and H874Y-AR than bicalutamide. Thus, RB346 is the first potent steroidal antiandrogen with efficacy for WT and various AR mutants.

Sodium thiosulfate pharmacokinetics in hemodialysis patients and healthy volunteers.

BACKGROUND AND OBJECTIVES: Vascular calcification is a major cause of morbidity and mortality in dialysis patients. Human and animal studies indicate that sodium thiosulfate (STS) may prevent the progression of vascular calcifications. The pharmacokinetics of STS in hemodialysis patients has not been investigated yet. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: STS was given intravenously to 10 hemodialysis patients on- and off-hemodialysis. Additionally, STS was applied to 9 healthy volunteers once intravenously and once orally. Thiosulfate concentrations were measured by using a specific and sensitive HPLC method. RESULTS: In volunteers and patients, mean endogenous thiosulfate baseline concentrations were 5.5 ± 1.82 versus 7.1 ± 2.7 µmol/L. Renal clearance was high in volunteers (1.86 ± 0.45 ml/min per kg) and reflected GFR. Nonrenal clearance was slightly, but not significantly, higher in volunteers (2.25 ± 0.32 ml/min per kg) than in anuric patients (2.04 ± 0.72 ml/min per kg). Hemodialysis clearance of STS was 2.62 ± 1.01 ml/min per kg. On the basis of the nonrenal clearance and the thiosulfate steady-state serum concentrations, a mean endogenous thiosulfate generation rate of 14.6 nmol/min per kg was calculated in patients. After oral application, only 4% of STS was recovered in urine of volunteers, reflecting a low bioavailability of 7.6% (0.8% to 26%). CONCLUSIONS: Given the low and variable bioavailability of oral STS, only intravenous STS should be prescribed today. The biologic relevance of the high hemodialysis clearance for the optimal time point of STS dosing awaits clarification of the mechanisms of action of STS.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice.

BACKGROUND: Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice. METHODS: First, DT-A plasmid transfection was evaluated, using the lipid formulation DMRIE-C in C4-2 prostate cancer xenografts. After detection of an overall high rate of apoptosis by DMRIE-C alone, plasmid delivery was performed in a second study by electroporation. Finally this method was used to specifically target the AR and PSA expressing cell line C4-2 using pDT-A driven by a prostate specific promoter and enhancer (PSE/PSA). PC-3 cells, being AR and PSA negative, served as controls. RESULTS: The experiments revealed evidence of a reduced growth rate of AR and PSA expressing C4-2 cells in vitro and in vivo compared to the AR and PSA negative prostate cancer cell line PC-3. The electroporation technology favored the response compared to DMRIE-C. CONCLUSION: These results suggest that the local delivery of DT-A plasmid by electroporation might present a favorable factor to treat prostate cancer.

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation.

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Regulation of placental growth by aldosterone and cortisol.

During pregnancy, trophoblasts grow to adapt the feto-maternal unit to fetal requirements. Aldosterone and cortisol levels increase, the latter being inactivated by a healthy placenta. By contrast, preeclamptic placental growth is reduced while aldosterone levels are low and placental cortisol tissue levels are high due to improper deactivation. Aldosterone acts as a growth factor in many tissues, whereas cortisol inhibits growth. We hypothesized that in preeclampsia low aldosterone and enhanced cortisol availability might mutually affect placental growth and function. Proliferation of cultured human trophoblasts was time- and dose-dependently increased with aldosterone (P < 0.04 to P < 0.0001) and inhibited by spironolactone and glucocorticoids (P < 0.01). Mineralo- and glucocorticoid receptor expression and activation upon agonist stimulation was verified by visualization of nuclear translocation of the receptors. Functional aldosterone deficiency simulated in pregnant mice by spironolactone treatment (15 µg/g body weight/day) led to a reduced fetal umbilical blood flow (P < 0.05). In rat (P < 0.05; R(2) = 0.2055) and human (X(2) = 3.85; P = 0.0249) pregnancy, placental size was positively related to plasma aldosterone. Autocrine production of these steroid hormones was excluded functionally and via the absence of specific enzymatic transcripts for CYP11B2 and CYP11B1. In conclusion, activation of mineralocorticoid receptors by maternal aldosterone appears to be required for trophoblast growth and a normal feto-placental function. Thus, low aldosterone levels and enhanced cortisol availability may be one explanation for the reduced placental size in preeclampsia and related disorders.

Urinary proteomics before and after extracorporeal circulation in patients with and without acute kidney injury.

OBJECTIVE: Acute kidney injury is a well-known complication with high morbidity and mortality after cardiopulmonary bypass. Cardiopulmonary bypass-associated acute kidney injury is still poorly understood. METHODS: Thirty-six patients undergoing elective cardiopulmonary bypass were enrolled. Spot urine samples before and after cardiopulmonary bypass were collected. Acute kidney injury was defined according to the RIFLE classification. To identify differentially regulated proteins after cardiopulmonary bypass, we first analyzed the urinary proteome before and after cardiopulmonary bypass. To identify differentially regulated proteins in acute kidney injury, we next compared the urinary proteome obtained on the first postoperative day between patients with and without acute kidney injury. Difference fluorescence gel electrophoresis was used to compare protein profiles and mass spectrometry to identify individual proteins. RESULTS: After cardiopulmonary bypass, inflammation-associated (zinc-alpha-2-glycoprotein, leucine-rich alpha-2-glycoprotein, mannan-binding lectin serine protease 2, basement membrane-specific heparan sulfate proteoglycan, and immunoglobulin kappa) or tubular dysfunction-associated (retinol-binding protein, adrenomedullin-binding protein, and uromodulin) proteins were differentially regulated. Acute kidney injury developed in 6 of 36 patients. A modified urinary albumin was increased, and zinc-alpha-2-glycoprotein and a fragment of adrenomedullin-binding protein were decreased in patients with acute kidney injury. Decreased excretion of zinc-alpha-2-glycoprotein in patients with acute kidney injury was confirmed by Western blot and enzyme-linked immunosorbent assay in an independent cohort of 22 patients with and 46 patients without acute kidney injury. CONCLUSION: Cardiopulmonary bypass leads to increased urinary excretion of inflammatory proteins and markers of tubular injury. Zinc-alpha-2-glycoprotein is a potentially useful predictive marker for acute kidney injury after cardiopulmonary bypass surgery.

'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex.

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.