A new method for QRS detection in ECG signals using QRS-preserving filtering techniques.

Detection of QRS complexes in ECG signals is required for various purposes such as determination of heart rate, feature extraction and classification. The problem of automatic QRS detection in ECG signals is complicated by the presence of noise spectrally overlapping with the QRS frequency range. As a solution to this problem, we propose the use of least-squares-optimisation-based smoothing techniques that suppress the noise peaks in the ECG while preserving the QRS complexes. We also propose a novel nonlinear transformation technique that is applied after the smoothing operations, which equalises the QRS amplitudes without boosting the supressed noise peaks. After these preprocessing operations, the R-peaks can finally be detected with high accuracy. The proposed technique has a low computational load and, therefore, it can be used for real-time QRS detection in a wearable device such as a Holter monitor or for fast offline QRS detection. The offline and real-time versions of the proposed technique have been evaluated on the standard MIT-BIH database. The offline implementation is found to perform better than state-of-the-art techniques based on wavelet transforms, empirical mode decomposition, etc. and the real-time implementation also shows improved performance over existing real-time QRS detection techniques.

Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates.

The steroid hydroxylases CYP17A1 (P450c17, 17-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) catalyze progesterone hydroxylation at one or more sites within a 2 Å radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[(2)H]-pregnenolone; 17-[(2)H]-, 16α-[(2)H]-, 21,21,21-[(2)H(3)]-, and 21-[(2)H]-progesterone; and 21,21,21-[(2)H(3)]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16α-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16α-hydroxylation) were used to calculate intramolecular and intermolecular kinetic isotope effects (KIEs). The intramolecular KIEs for CYP17A1 and mutation A105L were 4.1 and 3.8, respectively, at H-17 and 2.9 and 5.1, respectively, at H-16α. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16α-[(2)H]- and 17-[(2)H]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16α, respectively. Wild-type CYP21A2 also forms a trace of 16α-hydroxyprogesterone, which increased with 21,21,21-[(2)H(3)]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with (D)V values of 1.4-5.1 and (D)V/K values of 1.8-5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[(2)H]-progesterone with 33-44% deuterium retention, indicating stereochemical inversion. We conclude that human CYP17A1 has progesterone 21-hydroxylase activity and human CYP21A2 has progesterone 16α-hydroxylase activity, both of which are enhanced with deuterated substrates. The transition states for C-H bond cleavage in these hydroxylation reactions are either significantly nonlinear and/or asymmetric, and C-H bond breakage is partially rate-limiting for all reactions.

Abiraterone inhibits 3ß-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer.

PURPOSE: Treatment with abiraterone (abi) acetate prolongs survival in castration-resistant prostate cancer (CRPC). Resistance to abi invariably occurs, probably due in part to upregulation of steroidogenic enzymes and/or other mechanisms that sustain dihydrotestosterone (DHT) synthesis, which raises the possibility of reversing resistance by concomitant inhibition of other required steroidogenic enzymes. On the basis of the 3ß-hydroxyl, Δ(5)-structure, we hypothesized that abi also inhibits 3ß-hydroxysteroid dehydrogenase/isomerase (3ßHSD), which is absolutely required for DHT synthesis in CRPC, regardless of origins or routes of synthesis. EXPERIMENTAL DESIGN: We tested the effects of abi on 3ßHSD activity, androgen receptor localization, expression of androgen receptor-responsive genes, and CRPC growth in vivo. RESULTS: Abi inhibits recombinant 3ßHSD activity in vitro and endogenous 3ßHSD activity in LNCaP and LAPC4 cells, including conversion of [(3)H]-dehydroepiandrosterone (DHEA) to Δ(4)-androstenedione, androgen receptor nuclear translocation, expression of androgen receptor-responsive genes, and xenograft growth in orchiectomized mice supplemented with DHEA. Abi also blocks conversion of Δ(5)-androstenediol to testosterone by 3ßHSD. Abi inhibits 3ßHSD1 and 3ßHSD2 enzymatic activity in vitro; blocks conversion from DHEA to androstenedione and DHT with an IC(50) value of less than 1 µmol/L in CRPC cell lines; inhibits androgen receptor nuclear translocation; expression of TMPRSS2, prostate-specific antigen, and FKBP5; and decreases CRPC xenograft growth in DHEA-supplemented mice. CONCLUSIONS: We conclude that abi inhibits 3ßHSD-mediated conversion of DHEA to active androgens in CRPC. This second mode of action might be exploited to reverse resistance to CYP17A1 inhibition at the standard abi dose by dose-escalation or simply by administration with food to increase drug exposure.

Why human cytochrome P450c21 is a progesterone 21-hydroxylase.

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.

Identification of the nuclear receptor DAF-12 as a therapeutic target in parasitic nematodes.

Nematode parasitism is a worldwide health problem resulting in malnutrition and morbidity in over 1 billion people. The molecular mechanisms governing infection are poorly understood. Here, we report that an evolutionarily conserved nuclear hormone receptor signaling pathway governs development of the stage 3 infective larvae (iL3) in several nematode parasites, including Strongyloides stercoralis, Ancylostoma spp., and Necator americanus. As in the free-living Caenorhabditis elegans, steroid hormone-like dafachronic acids induced recovery of the dauer-like iL3 in parasitic nematodes by activating orthologs of the nuclear receptor DAF-12. Moreover, administration of dafachronic acid markedly reduced the pathogenic iL3 population in S. stercoralis, indicating the potential use of DAF-12 ligands to treat disseminated strongyloidiasis. To understand the pharmacology of targeting DAF-12, we solved the 3-dimensional structure of the S. stercoralis DAF-12 ligand-binding domain cocrystallized with dafachronic acids. These results reveal the molecular basis for DAF-12 ligand binding and identify nuclear receptors as unique therapeutic targets in parasitic nematodes.

Synthesis and activity of dafachronic acid ligands for the C. elegans DAF-12 nuclear hormone receptor.

The nuclear hormone receptor DAF-12 from Caenorhabditis elegans is activated by dafachronic acids, which derive from sterols upon oxidation by DAF-9, a cytochrome P450. DAF-12 activation is a critical checkpoint in C. elegans for acquisition of reproductive competence and for entry into adulthood rather than dauer diapause. Previous studies implicated the (25S)-Delta(7)-dafachronic acid isomer as the most potent compound, but the (25S)-Delta(4)-isomer was also identified as an activator of DAF-12. To explore the tolerance of DAF-12 for structural variations in the ligand and to enable further studies requiring large amounts of ligands for DAF-12 and homologs in other nematodes, we synthesized (25R)- and (25S)-isomers of five dafachronic acids differing in A/B-ring configurations. Both the (25S)- and (25R)-Delta(7)-dafachronic acids are potent transcriptional activators in a Gal4-transactivation assay using HEK-293 cells, with EC(50) values of 23 and 33 nm, respectively, as are (25S)- and (25R)-Delta(4)-dafachronic acids, with EC(50) values of 23 and 66 nm, respectively. The (25S)- and (25R)-Delta(5)-isomers were much less potent, with EC(50) values approaching 1000 nm, and saturated 5alpha- and 5beta-dafachronic acids showed mostly intermediate potencies. Rescue assays using daf- 9-null mutants confirmed the results from transactivation experiments, but this in vivo assay accentuated the greater potencies of the (25S)-epimers, particularly for the (25S)-Delta(7)-isomer. We conclude that DAF-12 accommodates a large range of structural variation in ligand geometry, but (25S)-Delta(7)-dafachronic acid is the most potent and probably biologically relevant isomer. Potency derives more from the A/B-ring configuration than from the stereochemistry at C-25.

Cofactors, redox state, and directional preferences of hydroxysteroid dehydrogenases.

The hydroxysteroid dehydrogenases (HSDs) interconvert pairs of weak and potent steroids, thus serving as key enzymes in the regulation of intracellular hormone potency. These enzymes may appear to drive unidirectional steroid flux in intact cells but actually catalyze bi-directional metabolism that achieve pseudo-equilibria with strong directional preferences. Even small shifts in the magnitude of these pseudo-equilibria can profoundly change steroid potency and thus contribute to disease. Consequently, we are studying the structural and biochemical principles that govern these directional preferences and the resilience of these pseudo-equilibria in intact cells. HSD directional preferences in intact cells are governed largely by relative affinities for nicotinamide cofactors [NAD(P)(H)] and existing cofactor gradients. We can attenuate the directional preferences for human 17betaHSD type 1 and rat AKR1C9 in intact cells by either diminishing the NADPH/NADP(+) gradient or by mutating the arginine residues that form salt bridges with the 2'-phosphate of NADP(H) (R38 and R276, respectively).

Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans.

In response to environmental and dietary cues, the C. elegans orphan nuclear receptor, DAF-12, regulates dauer diapause, reproductive development, fat metabolism, and life span. Despite strong evidence for hormonal control, the identification of the DAF-12 ligand has remained elusive. In this work, we identified two distinct 3-keto-cholestenoic acid metabolites of DAF-9, a cytochrome P450 involved in hormone production, that function as ligands for DAF-12. At nanomolar concentrations, these steroidal ligands (called dafachronic acids) bind and transactivate DAF-12 and rescue the hormone deficiency of daf-9 mutants. Interestingly, DAF-9 has a biochemical activity similar to mammalian CYP27A1 catalyzing addition of a terminal acid to the side chain of sterol metabolites. Together, these results define the first steroid hormones in nematodes as ligands for an invertebrate orphan nuclear receptor and demonstrate that steroidal regulation of reproduction, from biology to molecular mechanism, is conserved from worms to humans.

Deoxycorticosterone inactivation by AKR1C3 in human mineralocorticoid target tissues.

Aldosterone is the principal endogenous mineralocorticoid in humans and regulates salt and water homeostasis. Cortisol, the major glucocorticoid, has high affinity for the mineralocorticoid receptor; however, 11beta-hydroxysteroid dehydrogenase type 2 converts cortisol to the inactive steroid cortisone in aldosterone target cells of the kidney, thus limiting the mineralocorticoid action of cortisol. Deoxycorticosterone (DOC) binds to the mineralocorticocoid receptor with high affinity and circulates at concentrations comparable to aldosterone. Severe DOC excess as is seen in 17alpha- and 11beta-hydroxylase deficiencies causes hypertension, and moderate DOC overproduction in late pregnancy is associated with hypertension. Here, we demonstrate that DOC is inactivated by the 20-ketosteroid reductase activity of the human AKR1C3 isozyme. Immunohistochemical analyses demonstrate that AKR1C3 is expressed in the mineralocorticoid-responsive epithelial cells of the renal cortical and medullary collecting ducts, as well as the colon. Our findings suggest that AKR1C3 protects the mineralocorticoid receptor from activation by DOC in mineralocorticoid target cells of the kidney and colon, analogous to cortisol inactivation by 11beta-hydroxysteroid dehydrogenase type 2.

Stable 5,6-epoxyeicosatrienoic acid analog relaxes coronary arteries through potassium channel activation.

5,6-epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 micromol/L) induced concentration-dependent relaxations, with maximal relaxation of 86+/-5% and EC50 of 1 micromol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 micromol/L; max relaxation 43+/-9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 micromol/L; max relaxation 49+/-6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38+/-6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 micromol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46+/-13% and 52+/-7%, respectively; EC50 1micromol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether-containing prostaglandins that activate ATP-sensitive K+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.

Human 17beta-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells.

Human 17beta-hydroxysteroid dehydrogenases (17betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for 17betaHSD1 and 17beta HSD3 and only oxidative reactions for 17betaHSD2. We show that, after exhaustive incubations with either 17beta-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17betaHSD1 or 17betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three 17betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of 17betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that 17betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These 17betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.

Contributions of nitric oxide, EDHF, and EETs to endothelium-dependent relaxation in renal afferent arterioles.

BACKGROUND: Acetylcholine-induced endothelium-dependent relaxation in the renal afferent arteriole has been ascribed to nitric oxide, but the role of endothelium-derived hyperpolarizing factors (EDHFs) and 14,15-epoxyeicosatrienoic acid (14,15-EET) are unclear. METHODS: Single afferent arterioles were dissected from kidney of normal rabbits and microperfused in vitro at 60 mm Hg. Vessels were preconstricted submaximally with norepinephrine (10(-8) mol/L). Relaxation was assessed following cumulative addition of ACh (10(-9) to 10(-4) mol/L) alone, or in the presence of indomethacin (to inhibit cyclooxygenase), Nw-nitro-L-arginine (L-NNA) (to inhibit nitric oxide synthase), methylene blue (to inhibit soluble guanylate cyclase), or a combination of L-NNA + methylene blue. To assess contributions by EDHF, studies were repeated with either apamin + charybdotoxin [to block Ca2+-activated K+ channels (KCa)] or with 40 mmol/L KCl. To asses the role of 14,15-EET, relaxations were evaluated in the presence of its competitive inhibitor 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). RESULTS: Relaxation by acetylcholine was abolished following endothelial denudation. It was unaffected by indomethacin but was inhibited 54%+/- 5% (P < 0.001) by L-NNA, 57%+/- 5% by methylene blue, and 60%+/- 4% by the combination of L-NNA plus methylene blue. Relaxation was inhibited further by KCl (80%+/- 6%) or by apamin + charybdotoxin (96%+/- 2%). 14,15-EEZE, alone, inhibited acetylcholine-induced relaxation by 29%+/- 3%, and by 80%+/- 5% in the presence of L-NNA. CONCLUSION: Acetylcholine-induced afferent arteriolar relaxation depends strongly on both nitric oxide, acting via soluble guanylate cyclase, and on an EDHF, likely 14,15-EET, acting via KCa.

Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation.

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (ED(50) = 10(-6) M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a Delta8 double bond had full activity and potency. However, the analogs with only a Delta5 or Delta11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a Delta8 double bond, and a 14(S),15(R)-(cis)-epoxy group.