Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.

Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

Background: Vorapaxar is a new oral protease-activatedreceptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation. Methods: In this multinational, double-blind, randomized trial, we compared vorapaxar with placebo in 12,944 patients who had acute coronary syndromes without ST-segment elevation. The primary end point was a composite of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revascularization. RESULTS: Follow-up in the trial was terminated early after a safety review. After a median follow-up of 502 days (interquartile range, 349 to 667), the primary end point occurred in 1031 of 6473 patients receiving vorapaxar versus 1102 of 6471 patients receiving placebo (KaplanMeier 2-year rate, 18.5% vs. 19.9%; hazard ratio, 0.92; 95% confidence interval [CI], 0.85 to 1.01; P = 0.07). A composite of death from cardiovascular causes, myocardial infarction, or stroke occurred in 822 patients in the vorapaxar group versus 910 in the placebo group (14.7% and 16.4%, respectively; hazard ratio, 0.89; 95% CI, 0.81 to 0.98; P = 0.02). Rates of moderate and severe bleeding were 7.2% in the vorapaxar group and 5.2% in the placebo group (hazard ratio, 1.35; 95% CI, 1.16 to 1.58; P<0.001). Intracranial hemorrhage rates were 1.1% and 0.2%, respectively (hazard ratio, 3.39; 95% CI, 1.78 to 6.45; P<0.001). Rates of nonhemorrhagic adverse events were similar in the two groups. Conclusions: In patients with acute coronary syndromes, the addition of vorapaxar to standard therapy did not significantly reduce the primary composite end point but significantly increased the risk of major bleeding, including intracranial hemorrhage. (Funded by Merck; TRACER ClinicalTrials.gov number, NCT00527943.)

Acute coronary syndromes

Mehta et al. do not show any significant difference between early and delayed intervention in patients with acute coronary syndrome. In their study, 9.9% of patients in the...

Acute coronary syndromes

Mehta et al. do not show any significant difference between early and delayed intervention in patients with acute coronary syndrome. In their study, 9.9% of patients in the...

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

Bivalirudin for patients with acute coronary syndromes

Background: Current guidelines for patients with moderate- or high-risk acute coronary syndromes recommend an early invasive approach with concomitant antithrombotic therapy, including aspirin, clopidogrel, unfractionated or low-molecular-weight heparin, and glycoprotein IIb/IIIa inhibitors. We evaluated the role of thrombin-specific anticoagulation with bivalirudin in such patients. Methods: We assigned 13,819 patients with acute coronary syndromes to one of three antithrombotic regimens: unfractionated heparin or enoxaparin plus a glycoprotein IIb/IIIa inhibitor, bivalirudin plus a glycoprotein IIb/IIIa inhibitor, or bivalirudin alone. The primary end points were a composite ischemia end point (death, myocardial infarction, or unplanned revascularization for ischemia), major bleeding, and the net clinical outcome, defined as the combination of composite ischemia or major bleeding. Results: Bivalirudin plus a glycoprotein IIb/IIIa inhibitor, as compared with heparin plus a glycoprotein IIb/IIIa inhibitor, was associated with noninferior 30-day rates of the composite ischemia end point (7.7% and 7.3%, respectively), major bleeding (5.3% and 5.7%), and the net clinical outcome end point (11.8% and 11.7%). Bivalirudin alone, as compared with heparin plus a glycoprotein IIb/IIIa inhibitor, was associated with a noninferior rate of the composite ischemia end point (7.8% and 7.3%, respectively; P = 0.32; relative risk, 1.08; 95% confidence interval [CI], 0.93 to 1.24) and significantly reduced rates of major bleeding (3.0% vs. 5.7%; P<0.001; relative risk, 0.53; 95% CI, 0.43 to 0.65) and the net clinical outcome end point (10.1% vs. 11.7%; P = 0.02; relative risk, 0.86; 95% CI, 0.77 to 0.97). Conclusions: In patients with moderate- or high-risk acute coronary syndromes who were undergoing invasive treatment with glycoprotein IIb/IIIa inhibitors, bivalirudin was associated with rates of ischemia and bleeding that were similar to those with heparin. Bivalirudin alone was associated with similar rates of ischemia and significantly lower rates of bleeding. (ClinicalTrials.gov number, NCT00093158.)

Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

Background: Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation. Methods: In this multinational, double-blind, randomized trial, we compared vorapaxar with placebo in 12,944 patients who had acute coronary syndromes without ST-segment elevation. The primary end point was a composite of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revascularization. RESULTS: Follow-up in the trial was terminated early after a safety review. After a median follow-up of 502 days (interquartile range, 349 to 667), the primary end point occurred in 1031 of 6473 patients receiving vorapaxar versus 1102 of 6471 patients receiving placebo (Kaplan-Meier 2-year rate, 18.5% vs. 19.9%; hazard ratio, 0.92; 95% confidence interval [CI], 0.85 to 1.01; P = 0.07). A composite of death from cardiovascular causes, myocardial infarction, or stroke occurred in 822 patients in the vorapaxar group versus 910 in the placebo group (14.7% and 16.4%, respectively; hazard ratio, 0.89; 95% CI, 0.81 to 0.98; P = 0.02). Rates of moderate and severe bleeding were 7.2% in the vorapaxar group and 5.2% in the placebo group (hazard ratio, 1.35; 95% CI, 1.16 to 1.58; P<0.001). Intracranial hemorrhage rates were 1.1% and 0.2%, respectively (hazard ratio, 3.39; 95% CI, 1.78 to 6.45; P<0.001). Rates of nonhemorrhagic adverse events were similar in the two groups. Conclusions: In patients with acute coronary syndromes, the addition of vorapaxar to standard therapy did not significantly reduce the primary composite end point but significantly increased the risk of major bleeding, including intracranial hemorrhage. (Funded by Merck; TRACER ClinicalTrials.gov number, NCT00527943.)

Multiple supernumerary teeth not associated with complex syndromes: a retrospective study

Objectives: To determine the epidemiology and describe the clinical and radiographic characteristics, the type of treatment, and the possible delayed appearance of new supernumerary teeth in patients with non-syndromic multiple hyperdontia. Patients and Methods: We conducted a small retrospective observational study of 8 patients diagnosed with nonsyndromic multiple hyperdontia. Multiple hyperdontia not associated to complex syndromes was defined as apparently generally healthy patients with one or more supernumerary teeth in two or more areas. Results: The average patient age was 16.23 years; males predominated (3:1). Multiple hyperodontia with a minimum of 2 and a maximum of 9 supernumerary teeth was found (total: 34 mean: 4.25). The most frequent location was the upper jaw (76.47%). Eumorphic teeth were seen at lower premolar level, while the rest were all heteromorphic. There was altered eruption of the contiguous teeth of 4 of the impacted supernumerary teeth all the rest being asymptomatic. Extraction was the treatment in all patients, and in one of them the delayed appearance of 4 supernumerary teeth was detected. Conclusions: Multiple hyperodontia rarely occurs without being associated with complex syndromes. Prophylactic surgical removal of the supernumerary teeth is generally the treatment of choice.

Déficits y sesgos neurocognitivos y afectivos en la toma de decisiones de jugadores patológios y otros trastornos

Los déficits y sesgos tanto cognitivos como afectivos han sido fuente creciente de interés en el ámbito de la Neurociéncia de los Trastornos Mentales. En este proyecto, que se inicia en 2004 y finaliza a finales de 2008, se han estudiado los siguientes Trastornos Mentales: Juego Patológico (JP), Trastornos de la Conducta Alimentaria (TCA) y Trastornos Depresivos. En esta memoria nos centraremos en resumir parte de los resultados obtenidos en un estudio sobre JP y toma de decisiones (articulo en revisión y pendiente de aceptación) y otro de funcionamiento ejecutivo en JP y Bulimia Nerviosa (BN) (artículo en prensa). Resumiento el primer estudio los JP (N=32) muestran un proceso de toma de decisiones sesgado por la búsqueda de recompensa en forma de elevada toma de riesgos en comparación con Controles Sanos (CS). También se observan déficits en flexibilidad cognitiva pero no en control inhibitorio entre JP y CS. Los resultados descartan miopía conductual para lo toma de decisiones en JP, pero apuntan a un sesgo cognitivo-afectivo, en el que el control de los impulsos jugaría un papel relevante, en forma de ilusión de control, para los procesos de toma de decisiones con recompensa inmediata pero con castigo diferido, medidos por una prueba de toma de decisiones (IGT ABCD). En el segundo estudio, basándose en las vulnerabilidadades compartidas descritas entre JP y BN se comparó el funcionamiento ejecutivo de mujeres con JP y BN. Tras la administración del WCST y Stroop y ajustando el análisis por edad y educación, las JP mostraron mayor afectación, en concreto mayor porcentaje de errores perservaritvos, menor nivel de respuestas conceptuales y mayor número de ensayos administrados, mientras que el grupo de BN mostró mayor porcentaje de errores no persevarativos. Ambas, mujeres JP y BN mostraron disfunción ejecutiva en relación a los CS pero con diferentes correlatos específcos.

Cambios en los niveles de hormonas sexuales en el varón que envejece y su relación con el Síndrome Adam

En los últimos años está tomando un importante protagonismo el síndrome de ADAM (“Androgen Deficiency in Aging Males”), consecuencia del aumento de la expectativa de vida en los varones. Con la realización de este estudio pretendemos establecer la prevalencia de este síndrome en la población y establecer una relación entre los niveles androgénicos y el diagnóstico del síndrome de ADAM. Para ello escogeremos la muestra de la población de varones entre cincuenta y ochenta años del departamento 4 de Salud de la CCVV. La obtención de los datos se realizará mediante la realización de test clínicos fundamentalmente el cuestionario de ADAM.

Bases genètiques de la malformació de Chiari

La Malformació de Chiari tipus I (MCI) ha estat definida tradicionalment com la herniació de les amígdales cerebel•loses d’almenys 5mm, a través del forat mange. En general, els símptomes es posen de manifest durant la segona o tercera dècada de vida, tot i que s’han descrit casos pediàtrics. Donada la complexitat del quadre clínic, per realitzar un diagnòstic adient es requereix avaluació clínica i estudi de neuroimatge. La tècnica de preferència és la ressonància magnètica d’imatge, considerant-se actualment com a pacients de MCI aquells que presenten un descens de les amígdales superior a 3mm per sota del forat magne. L'existència de casos asimptomàtics dificulta establir una prevalença concreta, però s’ha estimat que podria estar entre 1/1000 a 1/5000 sent major en dones que en homes (2:1 aproximadament). Fins el moment, es desconeix l’etiologia de la malaltia però la hipòtesi més acceptada és que MCI és deguda al desenvolupament insuficient del mesoderm paraxial. Diferents estudis realitzats fins el moment evidencien que almenys, un subgrup de pacients amb MCI són deguts a contribució genètica: 1) casos d’agregació familiar amb afectes en tres generacions; 2) estudis de bessons 3) associació amb síndromes genètics coneguts amb herència mendeliana produïts per anomalies óssies que donen suport a la hipòtesi de la insuficiència del mesoderm com a causa de MCI. Davant l’evidència clara d’un component genètic com a principal causant de l’etiologia de MCI, l’objectiu del projecte va ser la identificació de les bases genètiques de la MCI, tant en gens responsables de les formes mendelianes com en gens responsables de les formes complexes de MCI mitjançant dues estratègies: 1-Identificació de variants genètiques de susceptibilitat en pacients amb MCI mitjançant estudis d’associació de tipus cas-control. 2-Anàlisi genètic de formes monogèniques mitjançant l’anàlisi de lligament a marcardors polimòrfics i la seqüenciació del DNA a gran escala.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

Expression cloning of a rat hepatic reduced glutathione transporter with canalicular characteristics

Using the Xenopus oocyte expression system, we have previously identified an approximately 4-kb fraction of mRNA from rat liver that expresses sulfobromophthalein-glutathione (BSP-GSH)-insensitive reduced glutathione (GSH) transport (Fernandez-Checa, J., J. R. Yi, C. Garcia-Ruiz, Z. Knezic, S. Tahara, and N. Kaplowitz. 1993. J. Biol. Chem. 268:2324-2328). Starting with a cDNA library constructed from this fraction, we have now isolated a single clone that expresses GSH transporter activity. The cDNA for the rat canalicular GSH transporter (RcGshT) is 4.05 kb with an open reading frame of 2,505 nucleotides encoding for a polypeptide of 835 amino acids (95,785 daltons). No identifiable homologies were found in searching various databases. An approximately 96-kD protein is generated in in vitro translation of cRNA for RcGshT. Northern blot analysis reveals a single 4-kb transcript in liver, kidney, intestine, lung, and brain. The abundance of mRNA for RcGshT in rat liver increased 3, 6, and 12 h after a single dose of phenobarbital. Insensitivity to BSP-GSH and induction by phenobarbital, unique characteristics of canalicular GSH secretion, suggest that RcGshT encodes for the canalicular GSH transporter.

Comparing 'visual' effect size indices for single-case designs

Effect size indices are indispensable for carrying out meta-analyses and can also be seen as an alternative for making decisions about the effectiveness of a treatment in an individual applied study. The desirable features of the procedures for quantifying the magnitude of intervention effect include educational/clinical meaningfulness, calculus easiness, insensitivity to autocorrelation, low false alarm and low miss rates. Three effect size indices related to visual analysis are compared according to the aforementioned criteria. The comparison is made by means of data sets with known parameters: degree of serial dependence, presence or absence of general trend, changes in level and/or in slope. The percent of nonoverlapping data showed the highest discrimination between data sets with and without intervention effect. In cases when autocorrelation or trend is present, the percentage of data points exceeding the median may be a better option to quantify the effectiveness of a psychological treatment.