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Francisca Campos,1 Javiera A Álvarez,1 Javiera Ortiz-Severín,1 Macarena A Varas,1 Carlos F Lagos,2 Ricardo Cabrera,3 Sergio A Álvarez,4 Francisco P Chávez11Laboratorio de Microbiología de Sistemas, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile; 2Chemical Biology & Drug Discovery Laboratory, Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago de Chile, Chile; 3Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile; 4Laboratorio de Microbiología, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas Y Farmacéuticas, Universidad de Chile, Santiago de Chile, ChileAbstract: Inorganic polyphosphate (polyP) and its metabolic enzymes are important in several cellular processes related with virulence and antibiotic susceptibility. Accordingly, bacterial polyP synthesis has been proposed as a good target for designing novel antivirulence molecules as alternative to conventional antibiotics. In most pathogenic bacteria, polyphosphate kinase 1 (PPK1), in charge of polyP synthesis from ATP, is widely conserved. Current colorimetric and radioactive polyP synthesis enzymatic assays are not suitable for high-throughput screening of PPK1 inhibitors. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4ʹ-6-diamidino-2-phenylindole), a fluorescence assay was previously developed by using a purified recombinant PPK1 enzyme from Escherichia coli. In this work we have developed a suitable methodology for high-throughput measurement of E. coli PPK1 activity. This platform can be used for the screening putative antimicrobial molecules for related enteropathogenic bacteria.Keywords: antivirulence, antimicrobial, affinity purification, polyphosphate kinase, DAPI, fluorescence enzymatic activity

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process., (© 2024. The Author(s).)

Temporal lobe epilepsy (TLE) is one of the most common types of epilepsy, yet approximately one-third of patients are refractory to current anticonvulsive drugs, which target neurons and synapses. Astrocytic and microglial dysfunction is commonly found in epileptic foci and has been shown to contribute to neuroinflammation and hyperexcitability in chronic epilepsy. Accumulating evidence points to a key role for glial hemichannels in epilepsy, but inhibiting both connexin (Cx) gap junctions and hemichannels can lead to undesirable side effects because the former coordinate physiological functions of cell assemblies. It would be a great benefit to use an orally available small molecule to block hemichannels to alleviate epileptic symptoms. Here, we explored the effect of D4, a newly developed compound that inhibits the Cx hemichannels but not Cx gap junctions using the pilocarpine mouse model of TLE. In vitro application of D4 caused a near-complete reduction in the pilocarpine-induced cell membrane permeability associated with increased Cx hemichannel activity. Moreover, preadministration of D4 in vivo effectively reduced neuroinflammation and altered synaptic inhibition, which then enhanced the animal survival rate. Posttreatment with a single dose of D4 in vivo has prolonged effects on suppressing the activation of astrocytes and microglia and rescued the changes in neuroinflammatory and synaptic gene expression induced by pilocarpine. Collectively, these results indicate that targeting Cx hemichannels by D4 is an effective and promising strategy for treating epilepsy in which neuroinflammation plays a critical role.

Ligand-based targeting of the receptors that are overexpressed explicitly on cancer cells represents an effective drug delivery approach to enhance the chemotherapeutic efficacy. Proprotein convertase subtilisin/kexin type 9 (PCSK9) which is a serine protease enzyme primarily produced by the liver cells, can potentially be used as a targeting ligand. PCSK9 binds to the LDL-r on hepatocytes' surface, leading to endocytosis and endosomal degradation. High LDL-r expression, which is believed to meet the higher demand of the cholesterol and phospholipids to build proliferating cancer cell membrane, ensures selective uptake of the PCSK9 conjugated liposomes. In the present work, the PCSK9 conjugated liposomal system was developed to deliver paclitaxel (PTX) to cancer cells. The protein was conjugated by EDC and NHS in a two-step coupling reaction to the liposomes containing COOH-PEG 2000 -COOH lipid. Conjugation was confirmed by NMR, and liposomes were further characterized by SEM and zeta sizer. PCSK9-conjugated liposomes showed high encapsulation efficiency of 69.1% with a diameter of 90.0 ± 4.9 nm. Long-term stability (30 days) study (Zeta potential: -9.88) confirmed excellent constancy and significant drug retention (58.2%). Invitro cytotoxicity and targeting efficiency was explored using MTS assay in human embryonic kidney cells (HEK293), liver hepatocellular cells (HEPG2), and a human colon cancer cell line (HCT116) for 24 h. PCSK9 conjugated liposomes exhibited significantly higher growth inhibition than the unconjugated (control) liposomes in HCT116 cell line (p < 0.001). The novel PCSK9 conjugated liposomes presented potent and precise in vitro anticancer activity and, therefore, are suggested for the first time as a promising targeted delivery system for cancer treatment., Competing Interests: Conflict of interest statement All Authors declare No Conflict of Interest., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Primary ovarian insufficiency (POI) affects 1% of women and carries significant medical and psychosocial sequelae. Approximately 10% of POI has a defined genetic cause, with most implicated genes relating to biological processes involved in early fetal ovary development and function. Recently, Ythdc2, an RNA helicase and N6-methyladenosine reader, has emerged as a regulator of meiosis in mice. Here, we describe homozygous pathogenic variants in YTHDC2 in 3 women with early-onset POI from 2 families: c. 2567C>G, p.P856R in the helicase-associated (HA2) domain and c.1129G>T, p.E377*. We demonstrated that YTHDC2 is expressed in the developing human fetal ovary and is upregulated in meiotic germ cells, together with related meiosis-associated factors. The p.P856R variant resulted in a less flexible protein that likely disrupted downstream conformational kinetics of the HA2 domain, whereas the p.E377* variant truncated the helicase core. Taken together, our results reveal that YTHDC2 is a key regulator of meiosis in humans and pathogenic variants within this gene are associated with POI.

Background: Familial hyperaldosteronism type I is caused by the generation of a chimeric aldosterone synthase enzyme (ASCE) which is regulated by ACTH instead of angiotensin II. We have reported that in vitro, the wild-type (ASWT) and chimeric aldosterone synthase (ASCE) enzymes are inhibited by progesterone and estradiol does not affect their activity., Aim: To explore the direct action of testosterone on ASWT and ASCE enzymes., Material and Methods: HEK-293 cells were transiently transfected with vectors containing the full ASWT or ASCE cDNAs. The effect of testosterone on AS enzyme activities was evaluated incubating HEK-cells transfected with enzyme vectors and adding deoxycorticosterone (DOC) alone or DOC plus increasing doses of testosterone. Aldosterone production was measured by HPLC-MS/MS. Docking of testosterone within the active sites of both enzymes was performed by modelling in silico., Results: In this system, testosterone inhibited ASWT (90% inhibition at five pM, 50% inhibitory concentration (IC50) =1.690 pM) with higher efficacy andpotency than ASCE (80% inhibition at five pM, IC50=3.176 pM). Molecular modelling studies showed different orientation of testosterone in ASWT and ASCE crystal structures., Conclusions: The inhibitory effect of testosterone on ASWT or ASCE enzymes is a novel non-genomic testosterone action, suggesting that further clinical studies are needed to assess the role of testosterone in the screening and diagnosis of primary aldosteronism.

A new series of twenty-two C-5 substituted N -arylsulfonylindoles was prepared with the aim of exploring the influence of C-5 substitution on 5-HT 6 receptor affinity. Eleven compounds showed moderate to high affinity at the receptor ( K i = 58-403 nM), with compound 4d being identified as the most potent ligand. However, regarding C-5 substitution, both methoxy and fluorine were detrimental for receptor affinity compared to our previously published unsubstituted compounds. In order to shed light on these observations, we performed docking and molecular dynamics simulations with the most potent compounds of each series ( 4d and 4l ) and PUC-10 , a highly active ligand previously reported by our group. The comparison brings about deeper insight about the influence of the C-5 substitution on the binding mode of the ligands, suggesting that these replacements are detrimental to the affinity due to precluding a ligand from reaching deeper inside the binding site. Additionally, CoMFA/CoMSIA studies were performed to systematize the information of the main structural and physicochemical characteristics of the ligands, which are responsible for their biological activity. The CoMFA and CoMSIA models presented high values of q 2 (0.653; 0.692) and r 2 (0.879; 0.970), respectively. Although the biological activity of the ligands can be explained in terms of the steric and electronic properties, it depends mainly on the electronic nature.

Introduction: Mineralocorticoid receptor (MR) activation within adipose tissue, triggers inflammation and metabolic syndrome development. The pharmacological blockade of MR provides beneficial effects for adipose tissue. Our study evaluates the impact of eplerenone implantation upon obesity. Experimental approach: A group of mice with implanted placebo pellets were fed using two types of diet, a normal (ND) or a high fat (HFD) diet. Additionally, a group of mice fed HFD were implanted with an eplerenone pellet. Metabolic and biochemical parameters were assessed in each animal group. Adipocyte size and lipid accumulation were investigated in the liver and adipose tissue. We evaluated the components of renin-angiotensin-aldosterone system (RAAS) locally in adipose tissue. Key results: Eplerenone reduced HFD-induced body weight gain, fasting glucose levels, fat accumulation, HFD-induced adipocyte size and liver lipid accumulation and improved glucose tolerance. In the adipose tissue, HFD significantly increased the mRNA levels of the RAAS molecules relative to the ND group. Eplerenone lowered RAAS mRNA levels, components of lipid metabolism and markers of inflammation in HFD-fed animals. Conclusion: MR antagonism with eplerenone diminishes insulin resistance that is related to obesity partly via a reduction of RAAS activation, inflammatory progression and cytokines induction. This suggests that eplerenone should be further studied as a therapeutic option for obesity and overweight., (Copyright © 2020 Vecchiola, Fuentes, Solar, Lagos, Opazo, Muñoz-Durango, Riedel, Owen, Kalergis and Fardella.)

Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.

The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73-93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (µ), chemical hardness (η), and electrophilicity (ω) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29-31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC 50 ) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa., Competing Interests: The authors declare no conflict of interest.

Purpose: 21-hydroxylase deficiency (21-OHD) is a congenital adrenal disease with more than 200 mutations published to date. The aim of this report is to describe a severe novel mutation of the CYP21A2 gene., Method: We describe a case of a 39-year-old male diagnosed with a salt wasting congenital adrenal hyperplasia (SWCAH) due to 21-OHD. The genetic testing was done using a combination of three methods (PCR XL, SALSA-MLPA, and bidirectional sequencing) and finally an in silico analysis., Results: The genetic testing demonstrated three severe mutations of the CYP21A2 gene (p.Gln318*; c.290-13C>G; and p.Trp86*), being the last one a novel mutation not previously reported. The in silico modeling of the p.Trp86* (c.258G>A) showed a truncated CYP21A2 protein that loses all the main structural features required for activity, such as the HEM binding domain and the hormone binding site., Conclusion: We present an adult man with an SWCAH due to 21-OHD who carried three severe mutations of the CYP21A2 gene, one of them, p.Trp86* (c.258G>A) has not been previously described.

Context: The genetic basis of human sex development is slowly being elucidated, and >40 different genetic causes of differences (or disorders) of sex development (DSDs) have now been reported. However, reaching a specific diagnosis using traditional approaches can be difficult, especially in adults where limited biochemical data may be available., Objective: We used a targeted next-generation sequencing approach to analyze known and candidate genes for DSDs in individuals with no specific molecular diagnosis., Participants and Design: We studied 52 adult 46,XY women attending a single-center adult service, who were part of a larger cohort of 400 individuals. Classic conditions such as17 β -hydroxysteroid dehydrogenase deficiency type 3, 5 α -reductase deficiency type 2, and androgen insensitivity syndrome were excluded. The study cohort had broad working diagnoses of complete gonadal dysgenesis (CGD) (n = 27) and partially virilized 46,XY DSD (pvDSD) (n = 25), a group that included partial gonadal dysgenesis and those with a broad "partial androgen insensitivity syndrome" label. Targeted sequencing of 180 genes was undertaken., Results: Overall, a likely genetic cause was found in 16 of 52 (30.8%) individuals (22.2% CGD, 40.0% pvDSD). Pathogenic variants were found in sex-determining region Y (SRY; n = 3), doublesex and mab-3-related transcription factor 1 (DMRT1; n = 1), NR5A1/steroidogenic factor-1 (SF-1) (n = 1), and desert hedgehog (DHH; n = 1) in the CGD group, and in NR5A1 (n = 5), DHH (n = 1), and DEAH-box helicase 37 (DHX37; n = 4) in the pvDSD group., Conclusions: Reaching a specific diagnosis can have clinical implications and provides insight into the role of these proteins in sex development. Next-generation sequencing approaches are invaluable, especially in adult populations or where diagnostic biochemistry is not possible., (Copyright © 2019 Endocrine Society.)

Background: Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations., Methods: An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants., Results: LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband's plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels., Conclusion: Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency.

Context: Classical apparent mineralocorticoid excess (AME) is a rare recessive disorder, caused by severe 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) deficiency. AME manifests as low-renin pediatric hypertension, hypokalemia and high cortisol/cortisone (F/E) ratio., Objective: To evaluate nonclassic AME (NC-AME) due to partial 11β-HSD2 insufficiency and its association with hypertension, mineralocorticoid receptor (MR) activation, and inflammatory parameters., Design: Cross-sectional study., Setting: Primary care cohort., Participants: We recruited 127 adolescents and adults. Subjects with secondary hypertension were excluded. We measured clinical, biochemical, renal, vascular, and inflammatory variables. Sequencing of HSD11B2 gene was performed in all subjects., Main Outcome Measure: NC-AME., Results: Serum F/E ratio was positively associated with systolic blood pressure (BP), microalbuminuria, and high-sensitivity C-reactive protein (hs-CRP). Serum cortisone correlated with MR activation parameters even when adjusted for age, body mass index, and sex: lower cortisone with higher potassium excretion (partial r = -0.29, P = 0.002) and with lower plasma renin activity (PRA) (partial r = 0.29, P = 0.001). Consistently, we identified 9 in 127 subjects (7.1%) with high F/E ratios (first quartile) and low cortisone (last quartile), suggestive of NC-AME. These subjects had higher systolic BP, 141.4 ± 25.7 mm Hg vs 127.3 ± 18.1 mm Hg, P = 0.03; lower PRA, 0.36 ± 0.19 ng/L*s vs 0.64 ± 0.47 ng/L*s, P < 0.0001; and greater potassium excretion, microalbuminuria, hs-CRP, and plasminogen activator inhibitor. We only found in 2 out of 9 subjects with NC-AME heterozygous mutations in the HSD11B2 gene., Conclusions: These findings suggest a spectrum of partial 11β-HSD2 insufficiency in a primary care cohort without the classic phenotype and genotype of AME. NC-AME may represent a phenotype of MR activation and cardiovascular risk, suggesting that these subjects could be treated with MR antagonists.

Background: Mounting evidence has associated high sodium (HS) intake with hypertension, cardiovascular disease, and stroke. We investigated whether HS intake modulates the parameters of endothelial damage, inflammation, and oxidative stress., Methods: We used a cross-sectional study design including 223 Chilean subjects (6.9-65.0 years old). We measured aldosterone, renin activity, cortisol, cortisone, adiponectin, leptin, hsCRP, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor type 1 (PAI-1), metalloproteinase (MMP)-9 and MMP-2 activity, and malondialdehyde. Sodium and creatinine were measured in 24-hour urine samples. The subjects were divided by sodium intake, high sodium (HS): ≥150 mEq/day, n = 118, and adequate sodium (AS): <150 mEq/day, n = 105., Results: We observed a positive correlation between urinary sodium excretion and blood pressure (r = 0.1669, P = 0.0124 for systolic and r = 0.2416, P = 0.0003 for diastolic), glycemia (r = 0.2660, P < 0.0001), and triglycerides (r = 0.1604, P = 0.0175) and a highly significant correlation between sodium excretion and PAI-1 (r = 0.2701, P < 0.0001). An inverse correlation was observed between urinary sodium and HDL-cholesterol (r = -0.2093, P = 0.0018) and adiponectin (r = -0.2679, P < 0.0001). In a linear regression model, urinary sodium excretion remained significantly associated with PAI-1 values even after adjusting for age, gender, and BMI. The HS group had higher blood pressure, glycemia, HOMA-IR, atherogenic index of plasma, and PAI-1 values than the group with AS intake., Conclusions: HS intake is associated with endothelial damage (high PAI-1) and metabolic dysregulation. On the other hand, inflammation and oxidative stress parameters are not modified by sodium intake.

Background: Pathogenic variations in HSD11B2 gene triggers the apparent mineralocorticoid excess syndrome (AME). There is scarce information regarding the phenotypes of subjects carrying heterozygous pathogenic variants in HSD11B2 gene. We investigated if serum cortisol/cortisone (F/E) ratio and cortisone are useful for identifying partial 11βHSD2 deficiency in those heterozygous subjects., Methods: We studied two patients diagnosed with AME and their families carrying either D223N or R213C mutation. We also evaluated 32 healthy control subjects (13 children and 19 adults) to obtain normal references ranges for all measured variables. Case 1: A boy carrying D223N mutation in HSD11B2 gene and Case 2: A girl carrying R213C mutation. We assessed serum F/E ratio and cortisone by HPLC-MS/MS, aldosterone, plasma-renin-activity(PRA), electrolytes, and HSD11B2 genetic analyses., Results: The normal values (median [interquartile range]) in children for serum F/E and cortisone (µg/dl) were 2.56 [2.21-3.69] and 2.54 [2.35-2.88], and in adults were 4.42 [3.70-4.90] and 2.23 [1.92-2.57], respectively. Case 1 showed a very high serum F/E 28.8 and low cortisone 0.46 µg/dl. His mother and sister were normotensives and heterozygous for D223N mutation with high F/E (13.2 and 6.0, respectively) and low cortisone (2.0 and 2.2, respectively). Case 2 showed a very high serum F/E 175 and suppressed cortisone 0.11 µg/dl. Her parents and sister were heterozygous for the R213C mutation with normal phenotype, but high F/E and low cortisone. Heterozygous subjects showed normal aldosterone, PRA, but lower fractional excretion of sodium and urinary Na/K ratio than controls., Conclusion: Serum F/E ratio and cortisone allow to identify partial 11βHSD2 deficiencies, as occurs in heterozygous subjects, who would be susceptible to develop arterial hypertension.

The corticotropin-releasing factor (CRF) system is a key mediator of the stress response and addictive behavior. The CRF system includes four peptides: The CRF system includes four peptides: CRF, urocortins I-III, CRF binding protein (CRF-BP) that binds CRF with high affinity, and two class B G-protein coupled receptors CRF 1 R and CRF 2 R. CRF-BP is a secreted protein without significant sequence homology to CRF receptors or to any other known class of protein. Recently, it has been described a potentiation role of CRF-BP over CRF signaling through CRF 2 R in addictive-related neuronal plasticity and behavior. In addition, it has been described that CRF-BP is capable to physically interact specifically with the α isoform of CRF 2 R and acts like an escort protein increasing the amount of the receptor in the plasma membrane. At present, there are no available structures for CRF-BP or for full-length CRFR. Knowing and studying the structure of these proteins could be beneficial in order to characterize the CRF-BP/CRF 2α R interaction. In this work, we report the modeling of CRF-BP and of full-length CRF 2α R and CRF 2β R based on the recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human CRF 1 R, in addition with the resolved N-terminal extracellular domain of CRFRs. These models were further studied using molecular dynamics simulations and protein-protein docking. The results predicted a higher possibility of interaction of CRF-BP with CRF 2α R than CRF 2β R and yielded the possible residues conforming the interacting interface. Thus, the present study provides a framework for further investigation of the CRF-BP/CRF 2α R interaction.

Purpose: DAX-1 (NR0B1) is an orphan nuclear receptor, which plays a critical role in development and regulation of the adrenal gland and hypothalamo-pituitary-gonadal axis. Mutations in NR0B1 lead to adrenal hypoplasia congenita (AHC), hypogonadotropic hypogonadism (HH) and azoospermia in men. Presentation is typically with adrenal insufficiency (AI) during infancy or childhood. To date only eight cases/kindreds are reported to have presented in adulthood., Methods: We describe two new cases of men with DAX-1 mutations who presented in adulthood and who were diagnosed at a large University Hospital., Results: Case 1 presented with AI at 19 years. At 38 years he was diagnosed with HH. Detailed history revealed a brother diagnosed with AI at a similar age. Sequencing of the DAX-1 (NR0B1) gene revealed a heterozygous c.775T > C substitution in exon 1, which changes codon 259 from serine to proline (p.Ser259Pro). Case 2 was diagnosed with AI at 30 years. Aged 37 years he presented with HH and azoospermia. He was treated with gonadotropin therapy but remained azoospermic. Testicular biopsy showed maturational arrest and hypospermatogenesis. Analysis of the NR0B1 gene showed a heterozygous c.836C > T substitution in exon 1, resulting in a change of codon 279 from proline to leucine (p.Pro279Leu). This change alters the structure of the repression helix domain of DAX-1 and affects protein complex interactions with NR5A family members., Conclusions: We describe two missense mutations within the putative carboxyl-terminal ligand binding domain of DAX-1, presenting with AHC and HH in adulthood, from a single center. DAX-1 mutations may be more frequent in adults than previously recognized. We recommend testing for DAX-1 mutations in all adults with primary AI and HH or impaired fertility where the etiology is unclear.

Factor Xa (FXa), a vitamin K-dependent serine protease plays a pivotal role in the coagulation cascade, one of the most interesting targets for the development of new anticoagulants. In the present work, we performed a virtual screening campaign based on ligand-based shape and electrostatic similarity search and protein-ligand docking to discover novel FXa-targeted scaffolds for further development of inhibitors. From an initial set of 260,000 compounds from the NCI Open database, 30 potential FXa inhibitors were identified and selected for in vitro biological evaluation. Compound 5 (NSC635393, 4-(3-methyl-4 H -1,4-benzothiazin-2-yl)-2,4-dioxo- N -phenylbutanamide) displayed an IC 50 value of 2.02 nM against human FXa. The identified compound may serve as starting point for the development of novel FXa inhibitors., Competing Interests: The authors declare no conflict of interest.

The preceding years have brought an exponential increase in our understanding of the endocannabinoid system (ECS), including the knowledge of CB1 and CB2 cannabinoid receptors, endocannabinoids, and the enzymes that synthesize and degrade endocannabinoids. Among these ECS components CB2 receptors have been the subject of considerable attention, primarily due to their promising therapeutic potential to treat numerous pathologies while avoiding the adverse psychotropic effects that can accompany CB1 receptor-based therapies. Recently, our research group has reported a new series of non-cytotoxic benzo[d]imidazoles and benzo[b]thiophenes displaying high CB2/CB1 selectivity index. In order to investigate the structural requirements for CB2 ligands and to derive a predictive model that can be used for the design of novel selective CB2 ligands, a three-dimensional quantitative structure-activity relationship (3D-QSAR) study was performed on the above mentioned chemical series employing comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) techniques. The CoMFA and CoMSIA models displayed high external predictability (r pred 2 0.919 and 0.908) and good statistical robustness. Valuable information regarding the steric, electrostatic and hydrophobic properties of the molecules was obtained, and several modifications around both heterocycles were evaluated with the aim to generate new promising series of benzo[d]imidazoles and benzo[b]thiophenes derivatives displaying high CB2 selectivity and low toxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The β₃ adrenergic receptor is raising as an important drug target for the treatment of pathologies such as diabetes, obesity, depression, and cardiac diseases among others. Several attempts to obtain selective and high affinity ligands have been made. Currently, Mirabegron is the only available drug on the market that targets this receptor approved for the treatment of overactive bladder. However, the FDA (Food and Drug Administration) in USA and the MHRA (Medicines and Healthcare products Regulatory Agency) in UK have made reports of potentially life-threatening side effects associated with the administration of Mirabegron, casting doubts on the continuity of this compound. Therefore, it is of utmost importance to gather information for the rational design and synthesis of new β₃ adrenergic ligands. Herein, we present the first combined 2D-QSAR (two-dimensional Quantitative Structure-Activity Relationship) and 3D-QSAR/CoMSIA (three-dimensional Quantitative Structure-Activity Relationship/Comparative Molecular Similarity Index Analysis) study on a series of potent β₃ adrenergic agonists of indole-alkylamine structure. We found a series of changes that can be made in the steric, hydrogen-bond donor and acceptor, lipophilicity and molar refractivity properties of the compounds to generate new promising molecules. Finally, based on our analysis, a summary and a regiospecific description of the requirements for improving β₃ adrenergic activity is given.

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca 2+ . Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Background: High sodium intake has been associated with various noncommunicable disease like hypertension, cardiovascular disease, or stroke. To estimate accurately sodium intake is challenging in clinical practice. We investigate the usefulness and limitations of assessing sodium intake simultaneously by dietary assessment and urinary samples in both children and adults., Methods: We used a cross-sectional study design inviting 298 Chilean subjects (74 children and 222 adults) aged between 9 and 66 years of both genders. Sodium intake by dietary assessment was obtained from Chilean food composition data, based on FAO tables. Sodium and creatinine excretion were measured in 24-hour urine samples, in all participants., Results: Adequate urinary collection was obtained in 81% of children (59/74) and 61% of adults (135/222). The mean sodium intake by dietary assessment was similar to the sodium excretion in 24 hours (3,121±1,153mg/d vs. 3,114±1,353mg/24h, P = nonsignificant) in children but was significantly lower (3,208±1,284mg/d vs. 4,160±1,651mg/24h, P < 0.001) in adults. In both children and adults, sodium intake correlated with urinary sodium excretion (r = 0.456, P < 0.003 and r = 0.390, P < 0.001, respectively). Secondary analyses also suggested that the dietary assessment was more inaccurate in overweight adult subjects., Conclusions: Our results showed that average sodium intake was higher than recommended in both children and adults (WHO ≤2,000mg/d). The sodium intake estimated by dietary assessment correlated with urinary excretion in all subjects, but in obese adults was more inaccurate than in children. Future studies to validate the appropriate test to assess sodium intake by age and nutritional status are warranted., (© American Journal of Hypertension, Ltd 2016. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To identify novel biomarkers associated with pediatric primary hypertension., Methods: We recruited 350 participants (4-16 years). Anthropometric parameters and aldosterone, plasma renin activity, cortisol, cortisone, Homeostasis Model Assessment Insulin Resistance (HOMA-IR), high-sensitivity C-reactive protein, adiponectin, IL-6, plasminogen activator inhibitor type 1 levels and matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9 and MMP-2) activities were measured. Genomic DNA was isolated. Patients with altered glucose metabolism, severe obesity [BMI-SD score (BMI-SDS) > 2.5], renovascular disease, primary aldosteronism and apparent mineralocorticoid excess syndrome were excluded., Results: In selected participants (n = 320), SBP was positively correlated with BMI-SDS (r = 0.382, P < 0.001), HOMA-IR (r = 0.211, P < 0.001), MMP-9 activity (r = 0.215, P < 0.001) and the cortisol/cortisone ratio (r = 0.231, P < 0.001). DBP showed similar correlations with these variables. No correlation was observed with aldosterone or plasma renin activity. Participants were categorized as hypertensive (n = 59) or nonhypertensive (n = 261). In the univariate analysis, hypertensive patients had higher BMI-SDS (P < 0.001), HOMA-IR (P < 0.001), high-sensitivity C-reactive protein (P < 0.001), MMP-9 activity (P < 0.001), plasminogen activator inhibitor type 1 (P < 0.001) and cortisol/cortisone ratio (P < 0.001) than nonhypertensive patients. Multiple regression analysis showed that the variables that remained associated with hypertension were higher BMI-SDS [odds ratio (OR) = 3.74; 95% confidence interval (CI) = 1.84-7.58], a higher cortisol/cortisone ratio (OR = 3.92; 95% CI = 1.98-7.71) and increased MMP-9 activity (OR = 4.23; 95% CI = 2.15-8.32)., Conclusion: We report that MMP-9 activity and the cortisol/cortisone ratio were higher in pediatric primary hypertensive patients, and these associations were independent of the effect of obesity. The potential role of these novel biomarkers in predicting hypertension risk and blood pressure regulation warrants further investigation.

Based on a known pharmacophore model for 5-HT₆ receptor antagonists, a series of novel extended derivatives of the N-arylsulfonyindole scaffold were designed and identified as a new class of 5-HT₆ receptor modulators. Eight of the compounds exhibited moderate to high binding affinities and displayed antagonist profile in 5-HT₆ receptor functional assays. Compounds 2-(4-(2-methoxyphenyl)piperazin-1-yl)-1-(1-tosyl-1H-indol-3-yl)ethanol (4b), 1-(1-(4-iodophenylsulfonyl)-1H-indol-3-yl)-2-(4-(2-methoxyphenyl)piperazin-1-yl)ethanol (4g) and 2-(4-(2-methoxyphenyl)piperazin-1-yl)-1-(1-(naphthalen-1-ylsulfonyl)-1H-indol-3-yl)ethanol (4j) showed the best binding affinity (4b pKi = 7.87; 4g pKi = 7.73; 4j pKi = 7.83). Additionally, compound 4j was identified as a highly potent antagonist (IC50 = 32 nM) in calcium mobilisation functional assay.

The interest of the pharmaceutical industry in developing new antibiotics is decreasing, as established screening systems which identify compounds that kill or inhibit the growth of bacteria can no longer be used. Consequently, antimicrobial screening using classical minimum inhibitory concentration (MIC) measurements is becoming obsolete. The discovery of antimicrobial agents that specifically target a bacterial pathogen without affecting the host and its beneficial bacteria is a promising strategy. However, few host-microbe models are available for in vivo screening of novel antivirulence molecules. Here we designed high-throughput developmental assays in the social amoeba Dictyostelium discoideum to measure Pseudomonas aeruginosa virulence and to screen for novel antivirulence molecules without side effects to the host and its beneficial bacteria Klebsiella aerogenes. Thirty compounds were evaluated that had been previously selected by virtual screening for inhibitors of P. aeruginosa PAO1 polyphosphate kinase 1 (PaPPK1) and diverse compounds with combined PPK1 inhibitory and antivirulence activities were identified. This approach demonstrates that D. discoideum is a suitable surrogate host for preliminary high-throughput screening of antivirulence agents and that PPK1 is a suitable target for developing novel antivirulence compounds that can be further validated in mammalian models., (Copyright © 2016 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

In the past decades, we have extended the view of aldosterone effects beyond epithelial tissues. New evidence regarding the aldosterone/mineralocorticoid receptor (MR) pathway in active metabolic tissues, including adipose tissue, has confirmed its pathogenic role in systemic inflammation, endothelial dysfunction, insulin resistance, and dyslipidemia. Obesity, a current epidemic worldwide, increases aldosterone production by several adipocyte factors such as leptin but is also associated with local aldosterone production. In addition, obesity can modulate MR activation leading to signaling dysregulation and a pro-inflammatory profile of adipocytes. Current knowledge have deciphered that this phenotypical differences of obesity may be explained, at least in part, by novel non-genomic activation of MR, new inducers of aldosterone synthesis, and probably by several epigenetic modifications. In addition, with the understanding of the complex interplay of obesity, hormones, and receptors, targeted pharmacological therapy is expected and is currently under active research.

Background: The impairment of 11β-hydroxysteroid dehydrogenase type 2 enzyme (11βHSD2) results in an inefficient conversion of cortisol to cortisone, which triggers hypertension. Cytosine-adenine repeat (CA repeat) microsatellite has been associated with low HSD11B2 gene expression., Aim: To determine whether the CA-repeat length in intron 1 affect the serum cortisol to cortisone (F/E) ratio and/or blood pressure (BP) levels in pediatric subjects., Subjects and Methods: Eighty-one hypertensive (HT) and 117 normotensive (NT) subjects participated in this study. We measured BP levels, as well as the F and E and F/E ratio in morning sera and 12-hour urine samples. The length of CA repeats was determined through fragment analysis. We compared the allele distribution between the HT and NT groups, and the patients were dichotomized into groups with short alleles (S) (<21 CA repeats) or long alleles (L), and also in groups according genotype (allele combination: S/S and S/L + L/L)., Results: We found no differences in the distribution of CA-repeat allelic length between the NT and HT groups (P = 0.7807), and there was no correlation between the CA-repeat allelic length and BP (P = 0.1151) levels or the serum F/E ratio (P = 0.6778). However, the serum F/E ratio was higher in the HT group than in the NT group (P = 0.0251). The serum F/E ratio was associated with systolic BP index independent of body mass index only in HT group., Conclusions: The CA-repeat length did not influence BP levels or serum F/E ratios in pediatric subjects. However, the serum F/E ratio was associated with BP, suggesting a role of 11βHSD2 in mineralocorticoid hypertension., (© American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid-base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS(812)LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI(623)LL and QVVA(635)LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI(623)LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI(623)LL with a highly conserved pocket in their γ1-σ1A hemicomplex., (© 2015 de la Fuente-Ortega, Gravotta, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Cerebrotendinous Xanthomatosis (CTX), a rare lipid storage disorder, is caused by recessive loss-of-function mutations of the 27-sterol hydroxylase (CYP27A1), producing an alteration of the synthesis of bile acids, with an accumulation of cholestanol. Clinical characteristics include juvenile cataracts, diarrhea, tendon xanthomas, cognitive impairment and other neurological manifestations. Early diagnosis is critical, because treatment with chenodeoxycholic acid may prevent neurological damage. We studied the CYP27A1 gene in two Chilean CTX patients by sequencing its nine exons, exon-intron boundaries, and cDNA from peripheral blood mononuclear cells. Patient 1 is a compound heterozygote for the novel substitution c.256-1G > T that causes exon 2 skipping, leading to a premature stop codon in exon 3, and for the previously-known pathogenic mutation c.1183C > T (p.Arg395Cys). Patient 2 is homozygous for the novel mutation c.1185-1G > A that causes exon 7 skipping and the generation of a premature stop codon in exon 8, leading to the loss of the crucial adrenoxin binding domain of CYP27A1.

A series of N-acyl-2,5-dimethoxyphenyl-1H-benzimidazoles were designed based on a CoMFA model for cannabinoid receptor type 1 (CB1) ligands. Compounds were synthesized and radioligand binding affinity assays were performed. Eight novel benzimidazoles exhibited affinity for the CB1 receptor in the nanomolar range, and the most promising derivative compound 5 displayed a K(i) value of 1.2 nM when compared to CP55,940. These results confirm our previously reported QSAR model on benzimidazole derivatives, providing new information for the development of small molecules with high CB1 affinity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chronic RAS (renin-angiotensin system) activation by both AngII (angiotensin II) and aldosterone leads to hypertension and perpetuates a cascade of pro-hypertrophic, pro-inflammatory, pro-thrombotic and atherogenic effects associated with cardiovascular damage. In 2000, a new pathway consisting of ACE2 (angiotensin-converting enzyme2), Ang-(1-9) [angiotensin-(1-9)], Ang-(1-7) [angiotensin-(1-7)] and the Mas receptor was discovered. Activation of this novel pathway stimulates vasodilation, anti-hypertrophy and anti-hyperplasia. For some time, studies have focused mainly on ACE2, Ang-(1-7) and the Mas receptor, and their biological properties that counterbalance the ACE/AngII/AT1R (angiotensin type 1 receptor) axis. No previous information about Ang-(1-9) suggested that this peptide had biological properties. However, recent data suggest that Ang-(1-9) protects the heart and blood vessels (and possibly the kidney) from adverse cardiovascular remodelling in patients with hypertension and/or heart failure. These beneficial effects are not modified by the Mas receptor antagonist A779 [an Ang-(1-7) receptor blocker], but they are abolished by the AT2R (angiotensin type 2 receptor) antagonist PD123319. Current information suggests that the beneficial effects of Ang-(1-9) are mediated via the AT2R. In the present review, we summarize the biological effects of the novel vasoactive peptide Ang-(1-9), providing new evidence of its cardiovascular-protective activity. We also discuss the potential mechanism by which this peptide prevents and ameliorates the cardiovascular damage induced by RAS activation.

11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone to cortisol in a NADPH dependent manner. Overexpression of 11β-HSD1 in key metabolic tissues is related to the development of type 2 diabetes, obesity, hypertension and metabolic syndrome. Using crystal structures of human 11β-HSD1 in complex with inhibitors as source of structural information, a combined ligand and structure-based virtual screening approach was implemented to identify novel 11β-HSD1 inhibitors. A selected group of compounds was identified in silico and further evaluated in cell-based assays for cytotoxicity and 11β-HSD1 mediated cortisol production inhibitory capacity. The expression of 11β-HSD1 and 11β-HSD2 in human LS14 adipocytes was assessed during differentiation. Biological evaluation of 39 compounds in adipocytes and steroids quantification by HPLC-MS/MS identify 4 compounds that exhibit 11β-HSD1 mediated cortisol production inhibitory activity with potencies in the micromolar range. Two compounds showed to be selective for the 11β-HSD1 reductase activity and over 11β-HSD2 isoform, and thus represent novel leads for the development of more active derivatives with higher efficacies targeting intracellular cortisol levels in type 2 diabetes and metabolic syndrome., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: The GTPase Rac1 has been implicated in hypertension as a modulator of mineralocorticoid receptor activity. Our aim is to investigate the frequency of polymorphisms rs10951982 (intron 1, G>A) and rs836478 (intron 3, T>C) in the RAC1 gene and perform association studies with clinical and biochemical parameters in a Chilean pediatric cohort., Methods: Two hundred two normotensive (NT) subjects (aged 4-16 years) were divided into 2 groups: NT subjects with hypertensive parents (NH; n = 103) and NT subjects with NT parents (NN; n = 99). We measured markers of inflammation (high-sensitivity C-reactive protein, interleukin 6 (IL-6), interleukin 8, and tumor necrosis factor α), endothelial damage (Plasminogen activator inhibitor-1 metalloproteinase-9, and metalloproteinase-2), and oxidative stress (malondialdehyde). Data were expressed as median and interquartile range (IQR)., Results: We found differences in polymorphism rs836478 (intron 3, C>T) in both genotypic (χ(2) = 15.2, 2 df; P = 0.0005) and allelic (X(2)=5.5, 1 df; P = 0.01) frequencies in NH vs. NN subjects. NH subjects with a TT genotype showed increase MMP9 expression (median = 2.3, IQR - 1.6-3.2; vs. median = 1.6, IQR = 1.6-2.3 AU; P = 0.01) and lower IL-6 expression (median = 8.8, IQR = 7.0-11.8; vs. median = 12.1, IQR = 8.2-14.7 pg/ml; P = 0.02) compared with subjects with TC/CC genotype. No difference in the allelic frequency distribution was seen in the polymorphism rs10951982 (NH vs. NN: χ(2)=0.2, 1 df; P = 0.6). For this SNP, NN subjects with GA/AA genotype showed decreased diastolic BP indexes compared with subjects with native GG genotype (median = 1.08, IQR = 1.0-1.2; vs. median = 0.99, IQR = 0.94-1.1; P = 0.02)., Conclusions: We report the frequency of polymorphisms rs836478 and rs10951982 of the RAC1 gene in a Spanish-Amerindian cohort. The polymorphism rs836478 was associated with an increased expression in markers of inflammation and endothelial damage (MMP9 and IL-6) in pediatric subjects with a hypertensive genetic background.

Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3β-hydroxyl (-OH) group on the cholestane ring to the common 3α-configuration on BAs. BAs are actively recaptured from the small intestine by the human Apical Sodium-dependent Bile Acid Transporter (hASBT) with high affinity and capacity. Previous studies have suggested that no particular hydroxyl group on BAs is critical for binding or transport by hASBT, even though 3β-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3α-OH group on BAs binding and translocation by hASBT. Ten 3β-hydroxylated BAs (Iso-bile acids, iBAs) were synthesized, characterized, and subjected to hASBT inhibition and uptake studies. hASBT inhibition and uptake kinetics of iBAs were compared to that of native 3α-OH BAs. Glycine conjugates of native and isomeric BAs were subjected to molecular dynamics simulations to identify topological descriptors related to binding and translocation by hASBT. Iso-BAs bound to hASBT with lower affinity and exhibited reduced translocation than their respective 3α-epimers. Kinetic data suggests that, in contrast to native BAs where hASBT binding is the rate-limiting step, iBAs transport was rate-limited by translocation and not binding. Remarkably, 7-dehydroxylated iBAs were not hASBT substrates, highlighting the critical role of 7-OH group on BA translocation by hASBT, especially for iBAs. Conformational analysis of gly-iBAs and native BAs identified topological features for optimal binding as: concave steroidal nucleus, 3-OH "on-" or below-steroidal plane, 7-OH below-plane, and 12-OH moiety toward-plane. Our results emphasize the relevance of the 3α-OH group on BAs for proper hASBT binding and transport and revealed the critical role of 7-OH group on BA translocation, particularly in the absence of a 3α-OH group. Results have implications for BA prodrug design.

Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by extreme reduction of white adipose tissue (WAT) mass. CGL type 1 is the most frequent form and is caused by mutations in AGPAT2. Genetic and clinical studies were performed in two affected sisters of a Chilean family. These patients have notoriously dissimilar metabolic abnormalities that correlate with differential levels of circulating leptin and soluble leptin receptor fraction. Sequencing of AGPAT2 exons and exon-intron boundaries revealed two homozygous mutations in both sisters. Missense mutation c.299G>A changes a conserved serine in the acyltransferase NHX4D motif of AGPAT2 (p.Ser100Asn). Intronic c.493-1G>C mutation destroy a conserved splicing site that likely leads to exon 4 skipping and deletion of whole AGPAT2 substrate binding domain. In silico protein modeling provided insights of the mechanisms of lack of catalytic activity owing to both mutations.

Cortisol homeostasis is implicated in hypertension and metabolic syndrome. Two enzymes modulate cortisol availability; 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) preferentially converts inactive cortisone to cortisol, whereas 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts cortisol to cortisone. In contrast, 5α and 5β reductases inactivate cortisol by conversion to its tetrahydrometabolites: tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone. A subtle local increase in cortisol can be detected by measuring 24-h urine metabolites, LC-MS/MS being the reference method. The 11β-HSD2 activity is assessed based on the cortisol/cortisone ratio, and the 11β-HSD1 activity on the (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ratio. To better understand hypertension and/or metabolic syndrome pathogenesis a method for simultaneous determination of cortisol, cortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone was developed and validated in an LC coupled with the new detector AB Sciex QTrap ® 4500 tandem mass spectrometer. The steroids were extracted from 1 mL urine, using cortisol-D4 as internal standard. The quantification range was 0.1-120 ng/mL for cortisol and cortisone, and 1-120 ng/mL for tetrahydrometabolites, with >89 % recovery for all analytes. The coefficient of variation and accuracy was <10 %, and 85-105 %, respectively. Our LC-MS/MS method is accurate and reproducible in accordance with Food and Drug Administration guidelines, showing good sensitivity and recovery. This method allows the assessment of 11β-HSD2 and 11β-HSD1 activities in a single analytical run providing an innovative tool to explain etiology of misclassified essential hypertension and/or metabolic syndrome.

Background: Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, resulting in the generation of a CYP11B1/B2 chimeric gene and abnormal adrenal aldosterone production. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Aldosterone levels rise during pregnancy, yet in pregnant women with FH-1, their hypertensive condition either remains unchanged or may even improve. The purpose of this study was to investigate in vitro whether female sex steroids modulate the activity of chimeric (ASCE) or wild type (ASWT) aldosterone synthase enzymes., Methods: We designed an in vitro assay using HEK-293 cell line transiently transfected with vectors containing the full ASCE or ASWT cDNAs. Progesterone or estradiol effects on AS enzyme activities were evaluated in transfected cells incubated with deoxycorticosterone (DOC) alone or DOC plus increasing doses of these steroids., Results: In our in vitro model, both enzymes showed similar apparent kinetic parameters (Km = 1.191 microM and Vmax = 27.08 microM/24 h for ASCE and Km = 1.163 microM and Vmax = 36.98 microM/24 h for ASWT; p = ns, Mann-Whitney test). Progesterone inhibited aldosterone production by ASCE- and ASWT-transfected cells, while estradiol demonstrated no effect. Progesterone acted as a competitive inhibitor for both enzymes. Molecular modelling studies and binding affinity estimations indicate that progesterone might bind to the substrate site in both ASCE and ASWT, supporting the idea that this steroid could regulate these enzymatic activities and contribute to the decay of aldosterone synthase activity in chimeric gene-positive patients., Conclusions: Our results show an inhibitory action of progesterone in the aldosterone synthesis by chimeric or wild type aldosterone synthase enzymes. This is a novel regulatory mechanism of progesterone action, which could be involved in protecting pregnant women with FH-1 against hypertension. In vitro, both enzymes showed comparable kinetic parameters, but ASWT was more strongly inhibited than ASCE. This study implicates a new role for progesterone in the regulation of aldosterone levels that could contribute, along with other factors, to the maintenance of an adequate aldosterone-progesterone balance in pregnancy.

A series of novel 2-pyridylbenzimidazole derivatives was rationally designed and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as a template for optimization. In the present series, 21 compounds displayed high affinities with Ki values in the nanomolar range. JM-39 (compound 39) was the most active of the series (KiCB1 = 0.53 nM), while compounds 31 and 44 exhibited similar affinities to WIN 55212-2. CoMFA analysis was performed based on the biological data obtained and resulted in a statistically significant CoMFA model with high predictive value (q2 = 0.710, r2 = 0.998, r2pred = 0.823).

Cocaine and Amphetamine Regulated Transcript (CART) peptides are anorexigenic neuropeptides. The L34F mutation in human CART peptide precursor (proCART) has been linked to obesity (Yanik et al. Endocrinology 147: 39, 2006). Decrease in CART peptide levels in individuals carrying the L34F mutation was attributed to proCART subcellular missorting. We studied proCART features required to enter the regulated secretory pathway. The subcellular localization and the secretion mode of monomeric EGFP fused to the full-length or truncated forms of human proCART transiently transfected in PC12 cells were analyzed. Our results showed that the N-terminal 1-41 fragment of proCART was necessary and sufficient to sort proCART to the regulated secretory pathway. In silico modeling predicted an alpha-helix structure located between residues 24-37 of proCART. Helical wheel projection of proCART alpha-helix showed an amphipathic configuration. The L34F mutation does not modify the amphipathicity of proCART alpha-helix and consistently proCARTL34F was efficiently sorted to the regulated secretory pathway. However, four additional mutations to proCARTL34F that reduced its alpha-helix amphipathicity resulted in the missorting of the mutated proCART toward the constitutive secretory pathway. These findings show that an amphipathic alpha-helix is a key cis-structure for the proCART sorting mechanism. In addition, our results indicate that the association between L34F mutation and obesity is not explained by proCART missorting.

Background: Severe hypertriglyceridemia (HTG) has been linked to defects in LPL, APOC2, APOA5, LMF1 and GBIHBP1 genes. However, a number of severe HTG cases are probably caused by as yet unidentified mutations. Very high triglyceride plasma levels (>112 mmol/L at diagnosis) were found in two sisters of a Chilean consanguineous family, which is strongly suggestive of a recessive highly penetrant mutation. The aim of this study was to determine the genetic locus responsible for the severe HTG in this family., Methods: We carried out a genome-wide linkage study with nearly 300,000 biallelic markers (Illumina Human CytoSNP-12 panel). Using the homozygosity mapping strategy, we searched for chromosome regions with excess of homozygous genotypes in the affected cases compared to non-affected relatives., Results: A large homozygous segment was found in the long arm of chromosome 11, with more than 2,500 consecutive homozygous SNP shared by the proband with her affected sister, and containing the APOA5/A4/C3/A1 cluster. Direct sequencing of the APOA5 gene revealed a known homozygous nonsense Q97X mutation (p.Gln97Ter) found in both affected sisters but not in non-affected relatives nor in a sample of unrelated controls., Conclusion: The Q97X mutation of the APOA5 gene in homozygous status is responsible for the severe hypertriglyceridemia in this family. We have shown that homozygosity mapping correctly pinpointed the genomic region containing the gene responsible for severe hypertriglyceridemia in this consanguineous Chilean family.

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.

Background: 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) inactivates cortisol (F) to cortisone (E); its impairment is associated with hypertension. We reported that 15.7% of the Chilean essential hypertensives possessed a high F/E ratio suggesting a partial deficit in 11β-HSD2 activity. It has been reported that the G534A(Glu178/Glu) polymorphism in the HSD11B2 gene is associated with hypertension. Investigate the frequency of the G534A polymorphism and its correlation with the glucocorticoid profile in Chilean essential hypertensive and normotensive subjects., Methods: Essential hypertensive outpatients (n = 232) and normotensive subjects (n = 74) were recruited. A change in the AluI restriction enzyme digest pattern, caused by the presence of the G534A polymorphism, was utilized to screen DNA isolated from leukocytes within the cohort before confirmation by sequencing. Plasma renin activity (PRA), serum aldosterone, F, and E were measured by radioimmunoassay. Urinary tetrahydrocortisol (THF), 5α-tetrahydrocortisol (5α-THF), and tetrahydrocortisone (THE) were measured by gas chromatography-mass spectrometry., Results: G534A polymorphism frequency was similar between hypertensive patients (19 of 232; 8.2%) and normotensive subjects (7 of 74; 9.5%). When categorized by presence or absence of the G534A polymorphism, no significant differences in the serum F/E ratio or other measured biochemical variables were detected. Despite a previous report that the G534A polymorphism is associated with a neighboring C468A (Thr156/Thr) polymorphism, analysis within our cohort showed that only one patient in each group presented with this double polymorphism., Conclusions: We report the frequency of the G534A polymorphism in the Spanish-Amerindian population. No correlation was detected between this polymorphism and the presence of hypertension and biochemical parameters in this Chilean cohort.

Trypanosoma cruzi glutamate cysteine ligase (TcGCL) is considered a potential drug target to develop novel antichagasic drugs. We have used a variety of computational methods to investigate the interactions between TcGCL with Glutathione (GSH). The three-dimensional structure of TcGCL was constructed by comparative modeling methods using the Saccharomyces cerevisiae glutamate cysteine ligase as template. Molecular dynamics simulations were used to validate the TcGCL model and to analyze the molecular interactions with GSH. Using RMSD clustering, the most prevalent GSH binding modes were identified paying attention to the residues involved in the molecular interactions. The GSH binding modes were used to propose pharmacophore models that can be exploited in further studies to identify novel antichagasic compounds.

Familial hyperaldosteronism type I is caused by an unequal crossover of 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes, giving rise to a chimeric CYP11B1/CYP11B2 gene (CG). We describe a family carrying a CG with high levels of free 18-hydroxycortisol but low prevalence of primary aldosteronism (PA) and an atypical CG inheritance pattern in a family of 4 generations with 16 adults and 13 children, we measured the arterial blood pressure, serum aldosterone, and plasma renin activity and then calculated the serum aldosterone:plasma renin activity ratio and urinary free 18-hydroxycortisol. We identified the CG by long-extension PCR and predicted its inheritance pattern. The CG was found in 24 of 29 subjects (10 children and 14 adults). In CG+ patients, hypertension and high 18-hydroxycortisol were prevalent (83% and 100%, respectively). High serum aldosterone:plasma renin activity ratio was more frequent in pediatric than adult patients (80% versus 36%; P<0.001). An inverse association between serum aldosterone:plasma renin activity ratio and age was observed (r=-0.48; P=0.018). Sequence analysis identified the CYP11B1/CYP11B2 crossover in a 50-bp region spanning intron 3 of CYP11B1 and exon 4 of CYP11B2. The CG segregation differs from an autosomal disease, showing 100% of CG penetrance in generations II and III. Statistical analysis suggests that inheritance pattern was not attributed to random segregation (P<0.001). In conclusion, we describe a family with an atypical CYP11B1/CYP11B2 gene inheritance pattern and variable phenotypic expression, where the majority of pediatric patients have primary aldosteronism. Most adults have normal aldosterone and renin levels, which could mask them as essential hypertensives.

Testicular injuries like torsion or cryptorchidism can cause massive germ cell death, which could have great impact on male reproductive health. In addition, it has been proposed that modern life style, in the form of underwear or sedentary work position, could increase the testicular temperature, induce germ cell apoptosis, reduce spermatozoa quality and promote male infertility. In this work we showed that a heat stress stimulus induced massive germ cells apoptosis, which was associated with p38 mitogen-activated protein kinase (MAPK) phosphorylation along with an increase in the levels of mRNA encoding calpain 2. Synthetic calpain inhibitors prevented heat stress-induced germ cell apoptosis through inhibition of p38 MAPK phosphorylation. Thus, our results indicate that the blockage of calpains suppresses p38 MAPK phosphorylation, and identifies calpain activation (most likely calpain 2) as an early event in heat stress-induced male germ cell apoptosis.