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BACKGROUND: Brain metastasis (BM) prognosis is incredibly poor and is often associated with considerable morbidity. Seizures are commonly present in these patients, and their biopsychosocial impact can be dangerous. The use of antiepileptic drugs (AEDs) as primary prophylaxis remains controversial. This systematic review and meta-analysis aim to evaluate the efficacy of AED prophylaxis in patients with BM. METHODS: MEDLINE via PubMed, Web of Science, EMBASE, and Cochrane were searched for articles pertinent to AED prophylaxis use in patients with BM. Patients with BM previously treated for cancer who were seizure naive at the time of inclusion were included. Data regarding patient characteristics, type of AED, prior treatments, and groups at a high risk of seizure were extracted. Seizure prevalence was obtained. RESULTS: Eight studies were included in this systematic review and meta-analysis; 1902 total patients with BM were included, with 381 receiving antiepileptic prophylaxis, and 1521 receiving no prophylaxis. Although the odds of a seizure in the treatment group was found to be 1.158 times the odds of a seizure in the control group, the odds ratio was not statistically significant (t-statistic = 0.62, P value = 0.5543). CONCLUSIONS: There was no significant difference in the odds of seizure development in control groups compared to patients receiving prophylactic antiepileptic therapy. As patients with BM present with heterogeneity in tumor characteristics and receive various treatment modalities, future research is needed to identify groups that may benefit more significantly from AED prophylaxis.
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Anticonvulsivantes , Neoplasias Encefálicas , Convulsiones , Humanos , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/complicaciones , Anticonvulsivantes/uso terapéutico , Convulsiones/prevención & control , Convulsiones/etiología , PrevalenciaRESUMEN
INTRODUCTION AND OBJECTIVE: Scholarly activity is a major component of residency training and the accreditation process for graduate medical education. In 2014, Accreditation Council for Graduate Medical Education and the American Association of Colleges of Osteopathic Medicine announced a single accreditation system with the transition beginning July 1, 2015. Previous data before the transition had shown that osteopathic physicians rarely published original research in three high-impact pediatric journals. The objective of this study is to determine if there is a degree disparity between osteopathic and allopathic physicians among authors who publish original research manuscripts in three high-impact pediatric journals after the beginning of the transition to a single graduate medical education accreditation system. Methods: Degree designation for the first and senior authors of original research manuscripts was reviewed for the Journal of Pediatrics (J Pediatr), Pediatrics, and JAMA Pediatrics (JAMA Pediatr) for the years 2016, 2017, 2018, and 2019. Inter-rater reliability was calculated by the kappa coefficient, and data were analyzed with descriptive statistics and simple linear regression. Results: A total of 3,252 manuscripts and 4,068 authors were reviewed with 0.98% of all authors being osteopathic physicians. A total of 1.65% of first authors and 0.41% of senior authors were osteopathic physicians. For those with a dual degree, a total of 1.03% of first, and 0.41% of senior authors were osteopathic physicians. No statistical trend could be established for increased first, senior, dual-degree first, or dual-degree senior osteopathic physician authorship. CONCLUSION: Osteopathic physicians continue to be underrepresented as first and senior authors in original publications in the three high-impact pediatric journals as compared to their allopathic counterparts.
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BACKGROUND: Minimally invasive lobectomy can be performed robotically or thoracoscopically. Short-term outcomes between the 2 approaches are reported to be similar; however, the comparative oncological effectiveness is not known. We sought to compare long-term survival after robotic and thoracoscopic lobectomy. METHODS: We performed a propensity-matched analysis of SEER (Surveillance, Epidemiology and End Results)-Medicare patients with non-small cell lung cancer from 2008 to 2013 who underwent minimally invasive lobectomy using either a thoracoscopic (n = 3881) or a robotic-assisted (n = 426) approach. Patients in the 2 groups were propensity matched 1:1 based on demographics, comorbidities, treatment, and tumor characteristics. We compared the overall survival (OS) and cancer-specific mortality (CSM) between the 2 groups. RESULTS: Within the matched cohort (n = 409 per group), the median age at surgery was 73 (range, 65-91) years, with a median follow-up of 35 months postsurgery. There was no difference in OS or CSM between the thoracoscopic and robotic-assisted groups (OS: 71.4% vs 73.1% at 3 years, overall P = .366; CSM: 16.6% vs 14.9% at 3 years, overall P = .639). CONCLUSIONS: Our propensity-matched analysis demonstrates that patients undergoing robotic-assisted lobectomy have similar OS and CSM compared with those patients undergoing thoracoscopic lobectomy. Oncologic outcomes are similar between the 2 minimally invasive approaches. These results demonstrate that further investigation is needed in the form of a randomized control trial, its variations, or additional large-scale registry analyses to verify these results.
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Carcinoma de Pulmón de Células no Pequeñas/cirugía , Neoplasias Pulmonares/cirugía , Neumonectomía/métodos , Puntaje de Propensión , Procedimientos Quirúrgicos Robotizados/métodos , Cirugía Torácica Asistida por Video/métodos , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , New Jersey/epidemiología , Estudios Retrospectivos , Tasa de Supervivencia/tendencias , Factores de TiempoRESUMEN
Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.
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Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento/métodos , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Temperatura , Línea Celular Tumoral , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary ß2 and ß3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either ß2 or ß3 subunits in HEK-293 cells. The ß2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the ß2 subunits did not change the biophysical properties of the channel. In contrast, ß3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated ß3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the ß3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the ß3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of ß subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with ß2. The ß3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.
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Activación del Canal Iónico , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Procesamiento Proteico-Postraduccional , Glicosilación , Células HEK293 , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Fosforilación , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
Androgen Receptor (AR) is a key driver in prostate cancer. Direct targeting of AR has valuable therapeutic potential. However, the lack of disease relevant cellular methodologies capable of discriminating between inhibitors that directly bind AR and those that instead act on AR co-regulators has made identification of novel antagonists challenging. The Cellular Thermal Shift Assay (CETSA) is a technology enabling confirmation of direct target engagement with label-free, endogenous protein in living cells. We report the development of the first high-throughput CETSA assay (CETSA HT) to identify direct AR binders in a prostate cancer cell line endogenously expressing AR. Using this approach, we screened a pharmacology library containing both compounds reported to directly engage AR, and compounds expected to target AR co-regulators. Our results show that CETSA HT exclusively identifies direct AR binders, differentiating them from co-regulator inhibitors where other cellular assays measuring functional responses cannot. Using this CETSA HT approach we can derive apparent binding affinities for a range of AR antagonists, which represent an intracellular measure of antagonist-receptor Ki performed for the first time in a label-free, disease-relevant context. These results highlight the potential of CETSA HT to improve the success rates for novel therapeutic interventions directly targeting AR.
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Ligandos , Receptores Androgénicos/metabolismo , Antagonistas de Receptores Androgénicos/metabolismo , Antagonistas de Receptores Androgénicos/farmacología , Andrógenos/metabolismo , Andrógenos/farmacología , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Transcripción GenéticaRESUMEN
Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs) has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1ß1δε, α3ß2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1ß1δε, α3ß2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.
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According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl(-) channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1ß. This aptamer potentiated whole-cell Cl(-) currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.
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Aptámeros de Nucleótidos/farmacología , Receptores de Glicina/agonistas , Animales , Aptámeros de Nucleótidos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Evaluación Preclínica de Medicamentos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Pichia , Receptores de Glicina/metabolismo , Técnica SELEX de Producción de Aptámeros , Resonancia por Plasmón de SuperficieRESUMEN
A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-ß (Aß) peptides in amyloid plaques. Aß peptides are produced by sequential cleavage of the amyloid precursor protein by the ß amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aß42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aß42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aß42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aß production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Sinergismo Farmacológico , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacología , Piranos/administración & dosificación , Piranos/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacologíaRESUMEN
The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.
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Canales de Calcio/química , Proteínas del Tejido Nervioso/química , Canales de Potencial de Receptor Transitorio/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio/genética , Canales de Calcio/metabolismo , Humanos , Canal de Potasio Kv.1.2/química , Canal de Potasio Kv.1.2/genética , Ligandos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oximas/farmacología , Mutación Puntual , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismo , XenopusRESUMEN
Congenital Insensitivity to Pain (CIP) is a loss of function mutation resulting in a truncated NaV1.7 protein, suggesting a pivotal role in pain signaling and rendering it an important pharmaceutical target for multiple pain conditions. The structural homology in the NaV-channel family makes it challenging to design effective analgesic compounds without inducing for example cardiotoxicity or seizure liabilities. An additional approach to structural isoform selectivity is to identify compounds with use- or state-dependent profiles, i.e. inhibition efficacy based on the gating of the ion channel. In general nerve cells in damaged or inflamed tissue are more depolarized and electrically active compared to healthy nerve cells in for instance the heart. This observation has led to the design of two types of screening protocols emulating the voltage condition of peripheral neurons or cardiac tissue. The two voltage protocols have been developed to identify both use- and state-dependent antagonists. In this paper we describe an attempt to merge the two different protocols into one to increase screening efficacy, while retaining relevant state- and use-dependent pharmacology. The new protocol is constructed of two stimulation pulses and a slow voltage ramp for simultaneous assessment of resting and state-dependent block. By comparing all protocols we show that the new protocol indeed filter compounds for state-dependence and increase the prediction power of selecting use-dependent compounds.
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Ensayos Analíticos de Alto Rendimiento/métodos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Células Cultivadas , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
AZD3043 (previously named THRX-918661) is a novel short-acting intravenous anesthetic agent in clinical trials. Although AZD3043 is a positive modulator at the γ-aminobutyric acid (GABA)(A)-receptor, its potency and efficacy have not been characterized in detail. Nor is it known whether the point-mutations in the ß-subunit of the GABA(A)-receptor that dramatically reduce the anesthetic effect of propofol (i.e. ß2 (N289M) and ß3 (N290M)), also influence the effect of AZD3043. This study investigated the in vitro pharmacology of AZD3043 at the most common human GABA(A) receptor subtypes. Subunits of four human wild-type (α1ß2, α1ß2γ2, α2ß2γ2 and α2ß3γ2) and two mutant (α1ß2(N289M)γ2 and α2ß3(N290M)γ2) GABA(A) receptor channels were introduced into Xenopus oocytes and studied with two-electrode voltage-clamp. AZD3043 potentiated and directly activated the α1ß2γ2, α2ß2γ2 and α2ß3γ2 GABA(A) receptor subtypes. Moreover, both potency and efficacy of AZD3043 were reduced at the mutant α1ß2(N289M)γ2 and α2ß3(N290M)γ2 subtypes. AZD3043 increased the GABA response also in GABA(A) receptors lacking the γ2-subunit, i.e. α1ß2. In conclusion, AZD3043 is a positive modulator and a direct agonist at human GABA(A) receptors and is not dependent on the γ2-subunit for its effect. Similar to propofol, the effect of AZD3043 is dramatically reduced by point-mutations in the ß2(N289M) and ß3(N290M) subunits, indicating similar molecular mechanisms of action for propofol and AZD3043 at the human GABA(A) receptor.
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Anestésicos/administración & dosificación , Anestésicos/farmacología , Fenilacetatos/administración & dosificación , Fenilacetatos/farmacología , Mutación Puntual , Receptores de GABA-A/metabolismo , Administración Intravenosa , Animales , Agonistas de Receptores de GABA-A/administración & dosificación , Agonistas de Receptores de GABA-A/farmacología , Humanos , Subunidades de Proteína/agonistas , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de GABA-A/genéticaRESUMEN
Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.
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Amidas/farmacología , Benzotiazoles/farmacología , Dolor/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Amidas/administración & dosificación , Amidas/química , Animales , Benzotiazoles/administración & dosificación , Benzotiazoles/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inflamación/tratamiento farmacológico , Estructura Molecular , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Solubilidad , Relación Estructura-ActividadRESUMEN
Fuculose-1-phosphate aldolase (FucA) is a useful biocatalyst with potential applications in chiral synthesis. In this study, the overall kinetic mechanism of FucA from the archaeon Methanococcus jannaschii was studied. The K(m) values of dihydroxyacetone phosphate (DHAP) and dl-glyceraldehyde were 0.09 and 0.74 mM, respectively. Dead-end inhibition by trimethyl phosphonoacetate and dl-threose were competitive and uncompetitive with respect to DHAP and dl-glyceraldehyde. Inhibition patterns obtained using reaction products were noncompetitive vs. DHAP and competitive vs. dl-glyceraldehyde. The equilibrium constant was 8.309×10(-3) M as assessed by varying the [DHAP]/[product] ratio at a fixed dl-glyceraldehyde concentration and by measuring the change in DHAP concentration after equilibrium was reached. This constant is consistent with the K(eq) value obtained from (13)C NMR (15.625×10(-3) M). The resultant inhibition kinetics may suggest the insights of kinetic mechanism of the FucA catalyzed reaction.
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Fructosa-Bifosfato Aldolasa/metabolismo , Hexosafosfatos/metabolismo , Methanococcus/enzimología , Proteínas Arqueales/metabolismo , Dihidroxiacetona Fosfato/química , Dihidroxiacetona Fosfato/metabolismo , Gliceraldehído/química , Gliceraldehído/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Especificidad por SustratoRESUMEN
4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 µM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. Caspase-3-dependent cleavage of the glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 h. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 h after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms.
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Aldehídos/toxicidad , Supervivencia Celular/efectos de los fármacos , Hepatocitos/enzimología , Aldehídos/química , Animales , Caspasa 3/metabolismo , Glutamato-Cisteína Ligasa/metabolismo , Hepatocitos/citología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , EstereoisomerismoRESUMEN
Pain-related problems among individuals in court-mandated Driver Intervention Programs (DIPs) for "driving under the influence" (DUI) offenders have not been well studied. This project examines 3,189 individuals from a DIP in Dayton, Ohio. Over 11% of participants reported significant pain-related interference in the past 4 weeks. Pain was significantly more likely in those with depression, more childhood conduct problems, and recent use of multiple illicit drugs. Many individuals seen in court-mandated DIP programs for DUI offenders also report difficulties with pain. DIP programming should address pain in relation to substance use and mental health issues.
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Intoxicación Alcohólica/epidemiología , Intoxicación Alcohólica/psicología , Conducción de Automóvil/legislación & jurisprudencia , Conducción de Automóvil/psicología , Dolor/epidemiología , Dolor/psicología , Prisioneros/legislación & jurisprudencia , Prisioneros/psicología , Adulto , Intoxicación Alcohólica/rehabilitación , Conducción de Automóvil/estadística & datos numéricos , Comorbilidad , Trastorno de la Conducta/epidemiología , Trastorno de la Conducta/psicología , Trastorno de la Conducta/rehabilitación , Estudios Transversales , Trastorno Depresivo/epidemiología , Trastorno Depresivo/psicología , Trastorno Depresivo/rehabilitación , Femenino , Humanos , Drogas Ilícitas , Masculino , Persona de Mediana Edad , Automedicación/psicología , Automedicación/estadística & datos numéricos , Factores Socioeconómicos , Centros de Tratamiento de Abuso de Sustancias , Trastornos Relacionados con Sustancias/epidemiología , Trastornos Relacionados con Sustancias/psicología , Trastornos Relacionados con Sustancias/rehabilitación , Encuestas y Cuestionarios , Adulto JovenRESUMEN
The polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants whose residues are increasing in fish, wildlife and human tissues. However, relatively little is known regarding the mechanisms of cell injury caused by PBDE congeners in fish. In the present study, we employed flow cytometry-based analyses to understand the onset and mechanisms of cell injury in rainbow trout gill cells (RTgill-W1 cells) exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47). Substantial optimization and validation for flow cytometry protocols were required during assay development for the trout gill cell line. Exposure to micromolar concentrations of BDE 47 elicited a significant loss in RTgill-W1 cell viability that was accompanied by a decrease in NAD(P)H autofluorescence, a marker associated with disruption of cellular redox status. This loss in NAD(P)H content was accompanied by a decrease in nonyl acridine orange fluorescence, indicating mitochondrial membrane lipid peroxidation. Furthermore, low doses of BDE 47 altered cellular forward angle light scatter (FS, a measure of cell diameter or size) and side light scatter properties (SS, a measure of cellular internal complexity), consistent with the early stages of apoptosis. These changes were more pronounced at higher BDE 47 concentrations, which led to an increase in the percentage of cells undergoing frank apoptosis as evidenced by sub-G1 DNA content. Apoptosis was also observed at a relatively low dose (3.2muM) of BDE 47 if cells were exposed for an extended period of time (24h). Collectively, the results of these studies indicate that exposure of rainbow trout gill cells to BDE47 is associated with the induction of apoptosis likely originating from disruption of cellular redox status and mitochondrial oxidative injury. The current report extends observations in other species demonstrating that oxidative stress is an important mechanism of BDE 47 mediated cellular toxicity, and supports the use of oxidative stress-associated biomarkers in assessing the sublethal effects of PBDEs and their replacements in fish. The application of flow cytometry endpoints using fish cell lines should facilitate study of the mechanisms of chemical injury in aquatic species.
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Células Epiteliales/efectos de los fármacos , Oncorhynchus mykiss/fisiología , Bifenilos Polibrominados/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Branquias/citología , Branquias/efectos de los fármacos , Éteres Difenilos Halogenados , Mitocondrias/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacosRESUMEN
The precise temperature control of the ABI Prism 7900HT Sequence Detection System designed for detection of fluorescence of a biological sample in real-time PCR assays (TaqMan assays) was used to activate Thermo-TRP ion channels, enabling a novel 384-/96-well plate-based assay. Functional pharmacology was verified against the temperature activation using intracellular calcium fluorescence as a measure of ion channel activity. The assay is applicable to both heterologous expression systems and dorsal root ganglia primary cells. This will benefit several analgesic drug discovery programs searching for new Thermo-TRP modulators.
Asunto(s)
Bioensayo/métodos , Células/metabolismo , Activación del Canal Iónico , Canales Catiónicos TRPV/metabolismo , Temperatura , Animales , Canales de Calcio/metabolismo , Línea Celular , Humanos , Proteínas del Tejido Nervioso/metabolismo , Ratas , Canal Catiónico TRPA1 , Canales Catiónicos TRPM/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
BACKGROUND: Nondepolarizing neuromuscular blocking agents (NMBAs) are classic competitive-inhibitors at the muscle nicotinic acetylcholine receptor (nAChR). Although the fetal subtype muscle nAChR has been extensively studied at a molecular level, less is known about the interaction between nondepolarizing NMBAs and the human adult muscle nAChR. The aim of this study was to investigate the effect of clinically used nondepolarizing NMBAs at human adult muscle nAChRs and the mechanisms behind the inhibition. METHODS: Human subunits for the adult alpha(1)beta(1)delta(epsilon) muscle nAChR were cloned and expressed into Xenopus oocytes and thereafter studied with two-electrode voltage clamp. The effect of the clinically used nondepolarizing NMBAs, including atracurium, cis-atracurium, mivacurium, pancuronium, rocuronium, vecuronium, and d-tubocurarine, on acetylcholine-induced and dimethylphenylpiperazinium-induced currents were investigated. RESULTS: All nondepolarizing NMBAs tested inhibited acetylcholine- and dimethylphenylpiperazinium-induced currents in human adult alpha(1)beta(1)delta(epsilon) muscle nAChRs, and no receptor activation was seen. Interestingly, acetylcholine desensitized the human adult alpha(1)beta(1)delta(epsilon) muscle type receptor and attenuated the inhibition caused by nondepolarizing NMBAs, as evident by lack of increase in IC(50) values for the nondepolarizing NMBAs with increased concentrations of acetylcholine. In contrast, dimethylphenylpiperazinium-induced currents were competitively inhibited by the nondepolarizing NMBAs. CONCLUSIONS: This study demonstrates that nondepolarizing NMBAs inhibit human adult muscle nAChRs expressed in Xenopus oocytes by mixed mechanisms. When using the nondesensitizing agonist dimethylphenylpiperazinium, inhibition by the NMBA is competitive, whereas activation with high concentrations of acetylcholine in combination with NMBA induces a noncompetitive inhibition, which the authors speculate can involve receptor desensitization similar to that observed in the neuromuscular junction.
Asunto(s)
Fármacos Neuromusculares no Despolarizantes/antagonistas & inhibidores , Receptores Nicotínicos/efectos de los fármacos , Acetilcolina/farmacología , Adulto , Algoritmos , Animales , Clonación Molecular , ADN/genética , Yoduro de Dimetilfenilpiperazina/farmacología , Electrofisiología , Humanos , Técnicas In Vitro , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Fármacos Neuromusculares no Despolarizantes/farmacología , Antagonistas Nicotínicos/farmacología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Receptores Nicotínicos/genética , Xenopus laevisRESUMEN
Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.