RESUMEN
BACKGROUND: Advances in lung cancer therapy have resulted in improved clinical outcomes. Unfortunately, advances can come at a financial cost to patients and their families that poses a significant risk to overall quality of life (QoL). Financial distress has been shown to be associated with increased symptom burden and decreased treatment compliance but the magnitude of financial distress is not well characterized in lung cancer populations. PATIENTS AND METHODS: Patients with stage II-IV newly diagnosed lung cancer and starting first-line therapy were recruited at a tertiary academic institution between July 2018 and April 2019. The comprehensive score for financial toxicity (COST) was used to assess financial toxicity and the Functional Assessment of Cancer Therapy-Lung (FACT-L) was used to assess QoL. Associations between financial toxicity and baseline variables were assessed using multivariable linear regression and correlations were assessed using the Pearson correlation. RESULTS: In this study, 143 consecutive patients were approached and 91.6% agreed to participate (N = 131). The median age was 65 years (35-90); 52.7% were male (n = 69), and 75.6% were white (n = 99). The inability to afford basic necessities and having <1 month of savings was associated with increased financial toxicity (P < 0.001) after adjusting for other factors such as age, race, insurance, and income. There was also a trend toward increased financial toxicity among those who were employed but on sick leave (P = 0.06). Increased financial toxicity was correlated with a decrease in QoL (correlation coefficient 0.41, P < 0.001). Patients' anticipated out-of-pocket (OOP) expenses for the upcoming 6 months ranged from $0 to $50 000 (median $2150). However, there was no correlation between anticipated OOP expenses and either financial toxicity or QoL. CONCLUSIONS: These data identify key factors for identifying at-risk patients and builds a framework for exploring the benefit of financial counseling interventions, which may improve QoL and oncologic outcomes.
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Neoplasias Pulmonares , Calidad de Vida , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Gastos en Salud , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , PercepciónRESUMEN
We investigate the temperature response of the alkali-metal rattling modes in ß-pyrochlores, AOs2O6 (A = K, Rb, Cs), from the results of ab initio molecular dynamics (MD) simulations performed at 20 K, 100 K and 300 K. Our results show that the temperature response of the T1u mode is clearly different from that of the T2g mode for all three pyrochlores. In this regard, two features are of particular note for both K and Rb; (1) the T1u mode exhibits a distinctly stronger softening response with decreasing temperature compared to the T2g mode, and (2) the T1u mode becomes stronger and sharper with decreasing temperature. These two findings suggest that the T1u mode is significantly more anharmonic and sensitive to the cage dynamics than the T2g mode. Examination of the local potentials around the alkali-metal atoms reveals that K has the flattest and most anharmonic potential at all temperatures while Cs exhibits the narrowest potential. The temperature dependence of the local potentials reveals that, for K, the potential at a higher temperature is not a simple extrapolation to higher energy of that at a lower temperature. Instead, we find significant reconstruction of the potential at different temperatures. Finally, we explore the temperature response of the coupling between the alkali metals and find a complex temperature dependence which suggests that the origin of the coupling may be more complex than a pure Coulomb interaction. We also find an unexpected increase in the static disorder of the system at low temperatures for the K and Rb pyrochlores.
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Metales Alcalinos/química , Modelos Químicos , Simulación de Dinámica Molecular , Niobio/química , Teoría Cuántica , Estructura Molecular , Temperatura , TermodinámicaRESUMEN
We have used ab initio molecular dynamics simulations validated against inelastic neutron scattering data to study alkali-metal dynamics in the ß-pyrochlore osmates AOs2O6 (A=K, Rb, Cs) at 300 K to gain insight into the microscopic nature of rattling dynamics in these materials. Our results provide new evidence at the microscopic level for rattling dynamics: (1) the elemental magnitude spectra calculated from the MD show a striking dominance by the alkali metals at low energies indicating weak coupling to the cage, (2) the atomic root-mean-square displacements for the alkali metals are significantly larger than for the other atoms, e.g., 25% and 150% larger than O and Os, respectively, in KOs2O6, and (3) motions of the alkali metals are weakly correlated to the dynamics in their immediate environment, e.g. K in KOs2O6 is 6 times less sensitive to its local environment than Os, indicating weak bonding of the K. There is broadening of the elemental spectra of the alkali metals from Cs to K corresponding to a similar broadening of the local potential around these atoms as determined from potential of mean-force calculations. This feature of the spectra is partly explained by the well-known increase in the relative cage volume with decreasing atomic size of the alkali metal. We find that for the smallest rattler in this series (K) the larger relative cage volume allows this atom freedom to explore a large space inside the cage leading to vibration at a broader range of frequencies, hence a broader spectrum. Thus, since K is considered the best rattler in this series, these findings suggest that a significant feature of a good rattler is the ability to vibrate at several different but closely spaced frequencies.
RESUMEN
At the Alaska Native Medical Center in Anchorage, colorectal cancer screening rates improved dramatically with the initiation of a dedicated flexible sigmoidoscopy screening program staffed by mid-level providers. We describe the development and implementation of a program to train rural nurse practitioners and physician assistants in flexible sigmoidoscopy.
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Neoplasias Colorrectales/diagnóstico , Detección Precoz del Cáncer/estadística & datos numéricos , Promoción de la Salud/métodos , Indígenas Norteamericanos , Enfermeras Practicantes/educación , Asistentes Médicos/educación , Sigmoidoscopía , Alaska , Servicios de Salud Comunitaria , Curriculum , Humanos , Indígenas Norteamericanos/psicología , Aceptación de la Atención de Salud , Relaciones Profesional-Paciente , Desarrollo de Programa , Servicios de Salud RuralRESUMEN
The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) is reported to have anticancer activity in vivo. Induction of cell cycle arrest and apoptosis in cancer cell lines refractory to standard retinoids suggests a retinoid-independent mechanism of action for AHPN. Conformational studies suggested that binding of AHPN does not induce an unusual conformation in retinoic acid receptor (RAR) gamma. The 3-chloro AHPN analogue MM11453 inhibited the growth of both retinoid-resistant (HL-60R leukemia, MDA-MB-231 breast, and H292 lung) and retinoid-sensitive (MCF-7 breast, LNCaP prostate, and H460 lung) cancer cell lines by inducing apoptosis at similar concentrations. Before apoptosis, MM11453 induced transcription factor TR3 expression and loss of mitochondrial membrane potential characteristic of apoptosis. MM11453 lacked the ability to significantly activate RARs and retinoid X receptor alpha to initiate (TREpal)(2)-tk-CAT reporter transcription. These results, differential proteolysis-sensitivity assays, and glutathione S-transferase-pulldown experiments demonstrate that, unlike AHPN or the natural or standard synthetic retinoids, MM11453 does not behave as a RAR or retinoid X receptor alpha transcriptional agonist. These studies strongly suggest that AHPN exerts its cell cycle arrest and apoptotic activity by a signaling pathway independent of retinoid receptor activation.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Retinoides/farmacología , Activación Transcripcional/efectos de los fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/biosíntesis , Inhibidores de Crecimiento/farmacología , Células HL-60 , Células HeLa , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Células Jurkat , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Conformación Molecular , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Conformación Proteica , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides , Retinoides/metabolismo , Factores de Transcripción/biosíntesisRESUMEN
We evaluated the use of a new lactate oxidase-based reagent for the determination of serum and plasma lactic acid levels with the Hitachi 911 (Roche Diagnostics, Indianapolis, IN) and the Beckman CX7 (Beckman Instruments, Brea, CA). Evaluation studies demonstrated on-board stability of at least 3 months and a calibration stability of more than 5 months. Within- and between-day imprecision of this reagent was less than 2% for both applications. The reagent is free of the deleterious effects of triglyceride up to levels of 1,400 mg/dL (15.8 mmol/L), bilirubin to concentrations of 24.6 mg/dL (420 mumol/L), and hemoglobin, from lysed erythrocytes, to levels of more than 0.3 g/dL (3.0 g/L). When used on the Hitachi 911 for the determination of plasma lactate concentrations, the reagent correlates with the Dade aca III (Dade International, Deerfield, IL). When applied to the Beckman CX7 for the determination of serum lactate levels, the method correlates with the Beckman method.
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Análisis Químico de la Sangre/instrumentación , Análisis Químico de la Sangre/métodos , Indicadores y Reactivos/química , Ácido Láctico/sangre , Ampirona , Calibración , Estabilidad de Medicamentos , Estudios de Evaluación como Asunto , Humanos , Indicadores y Reactivos/normas , Oxigenasas de Función Mixta , Concentración Osmolar , ToluidinasRESUMEN
The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL-60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9-cis-retinoic acid (9-cis-RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9-cis-RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA-MB-468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR-signaling pathways.
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Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Portadoras/aislamiento & purificación , Retinoides/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Femenino , Células HL-60 , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retinoides/farmacología , Receptor de Ácido Retinoico gammaRESUMEN
A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy. GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell. Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells. Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins. GRASP. GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane. Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.
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Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Tretinoina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Extractos Celulares , Clonación Molecular , ADN Complementario , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de la Membrana , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor alpha (PPARalpha), and PPARalpha ligands suppressed this interaction. In contrast to the interaction of PPARalpha with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARalpha ligand binding domain. NCoR was found to suppress PPARalpha-dependent transcriptional activation in the context of a PPARalpha.retinoid X receptor alpha (RXRalpha) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARalpha.RXRalpha heterodimeric complexes in mammalian cells. NCoR appears to influence PPARalpha signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.
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Microcuerpos/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Clonación Molecular , Dimerización , Humanos , Ligandos , Datos de Secuencia Molecular , Mutación/genética , Co-Represor 1 de Receptor Nuclear , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras/genética , Receptores X Retinoide , Transducción de Señal/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Transfección/genética , Levaduras/genética , Receptor de Ácido Retinoico gammaRESUMEN
Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.
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Proteínas Aviares , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides , Factores de Transcripción/metabolismo , Animales , Factor de Transcripción COUP I , Factor de Transcripción COUP II , Factores de Transcripción COUP , Línea Celular , Pollos , Dimerización , Humanos , Ovalbúmina/genética , Ovalbúmina/metabolismo , Unión Proteica , Proteínas RepresorasAsunto(s)
Vasos Sanguíneos/metabolismo , Proteínas Portadoras/biosíntesis , Células del Tejido Conectivo/metabolismo , Receptores de Antígenos de Linfocitos B/biosíntesis , Proteínas Adaptadoras Transductoras de Señales , Vasos Sanguíneos/citología , Proteínas de la Matriz Extracelular/biosíntesis , Técnica del Anticuerpo Fluorescente Indirecta , HumanosRESUMEN
The interaction of retinoid X receptor alpha with 9-cis-retinoic acid was studied using stopped-flow fluorescence spectroscopy. Transient kinetic analyses of this interaction suggest a two-step binding mechanism involving a rapid, enthalpically driven pre-equilibrium followed by a slower, entropically driven reaction that may arise from a conformational change within the ligand binding domain of the receptor. The assignment of this kinetic mechanism was supported by agreement between the overall equilibrium constant, Kov, derived from kinetic studies with that determined by equilibrium fluorescence titrations. Although these analyses do not preclude ligand-induced alteration in the oligomerization state of the receptor in solution, the simplest model that can be applied to these data involves the stoichiometric interaction of 9-cis-retinoic acid with retinoid X receptor alpha monomers.
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Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Tretinoina/química , Tretinoina/metabolismo , Alitretinoína , Animales , Sitios de Unión , Cromatografía en Gel , Cinética , Ligandos , Ratones , Conformación Proteica , Receptores X Retinoide , Espectrometría de Fluorescencia , Temperatura , Termodinámica , Triptófano/químicaRESUMEN
CATH.a cells are a catecholaminergic cell line of neuronal origin. The opioid receptor complement expressed by CATH.a cells was defined pharmacologically and by reverse transcription-polymerase chain reaction (RT-PCR). CATH.a cells were found to express mRNA encoding all three of the major subtypes of opioid receptors. The relative abundance of CATH.a cell opioid receptor transcripts was delta > kappa> mu. Pharmacological and functional data were in agreement with the results of RT-PCR inasmuch as delta-opioid receptor was identified as the most abundant opioid receptor subtype expressed by CATH.a cells. In addition, at least one of the opioid signalling pathways, inhibition of adenylyl cyclase activity, was found to be operant in this cell line. CATH.a cells should be of general utility for the study of opioid receptor signalling mechanisms in the context of catecholaminergic neurons.
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Catecolaminas/fisiología , Neuronas/metabolismo , Receptores sigma/biosíntesis , Adenilil Ciclasas/metabolismo , Animales , Línea Celular , Diprenorfina/metabolismo , Relación Dosis-Respuesta a Droga , Encefalina D-Penicilamina (2,5) , Encefalinas/farmacología , Guanilil Imidodifosfato/farmacología , Técnicas In Vitro , Ratones , Antagonistas de Narcóticos/metabolismo , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Receptores Opioides delta/agonistas , Receptores sigma/efectos de los fármacos , Receptores sigma/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Patient and staff ratings of the importance of caring behaviours (Caring Assessment Instrument, CARE-Q) were studied and related to ratings of patient levels of anxiety and depression (Hospital Anxiety and Depression Scale) in 53 cancer patient-staff dyads. Both groups perceived anticipatory and comforting behaviours to be among the three most important. Patients considered staff explanation and facilitation as well as anticipation to be more important than did staff. Staff rated accessibility and comforting as more important than did patients. Patient and staff ratings of the importance of staff accessibility were negatively correlated. Thus, patient and staff 'did not agree strongly on the importance of several types of caring behaviours. Neither patient nor staff ratings of the importance of caring behaviours were associated with their ratings of the levels of anxiety or depression of specific patients. The results suggest that patient-staff communication requires specific knowledge and skills to make staff accurately judge what is important in making patients feel cared for.
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Ansiedad/psicología , Actitud del Personal de Salud , Actitud Frente a la Salud , Depresión/psicología , Empatía , Neoplasias/enfermería , Neoplasias/psicología , Relaciones Enfermero-Paciente , Personal de Enfermería en Hospital/psicología , Adulto , Anciano , Anciano de 80 o más Años , Comunicación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Evaluación en Enfermería , Investigación Metodológica en Enfermería , Encuestas y CuestionariosRESUMEN
Neuroleptic drugs that bind sigma sites were tested for their ability to inhibit growth and radiosensitize MCF-7 human breast cancer cells. Inhibition of growth by approximately 50% occurred in cells exposed to pimozide (0.6 microM), haloperidol (10 microM), and the sigma ligand DTG (1,3-di(2-tolyl)guanidine, 20 microM), but no growth inhibition occurred in cells exposed to clozapine, a neuroleptic drug lacking sigma binding activity, or dextromethorphan, a selective sigma 1 binding ligand. Pimozide (2.5 microM), but not haloperidol (3.6 microM), enhanced the sensitivity of MCF-7 cells to gamma radiation in clonogenic survival assays. Pimozide significantly decreased MCF-7 clonogenic survival following a 5 or 8 Gy dose of gamma radiation, and the dose of radiation required for 1% survival (survival enhancement ratio, SER) was decreased by a factor of 2. Exposure of normal WI-38 human embryonic lung cells to pimozide did not increase their sensitivity to gamma radiation. Pimozide (2.5 microM) activated early apoptotic changes in MCF-7 cells that were detected by the uptake of Hoechst 33342 dye, and 10 microM pimozide activated a complete apoptotic pathway resulting in the death of > 90% of the cells within 24 hours. MCF-7 cells exposed to gamma radiation alone (8 Gy) showed giant cell formation, mitotic arrest, and a limited degree of apoptosis and necrosis. Within 50 hours of treatment with a combination of radiation and pimozide, cell numbers were sharply reduced compared with cultures exposed to either radiation or pimozide alone. We conclude that pimozide augmented the sensitivity of MCF-7 cells to radiation-induced cell killing through a mechanism not shared by haloperidol, but suggest that concentration of pimozide in MCF-7 cells as a result of an enrichment of sigma 2 sites might target the radiosensitization.
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Antipsicóticos/farmacología , Neoplasias de la Mama/patología , Rayos gamma , Pimozida/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Apoptosis , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Femenino , Citometría de Flujo , Guanidinas/farmacología , Haloperidol/farmacología , Humanos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
OBJECTIVE: To determine whether expression of neutrophil integrin receptors is related to the degree of post-traumatic shock. DESIGN: Data were collected prospectively on patients with major trauma admitted to the surgical intensive care unit. SETTING: Denver General Hospital, Colorado. PATIENTS AND PARTICIPANTS: 17 severely injured adults. MEASUREMENTS AND RESULTS: The mean fluorescence intensity and per cent positive of neutrophil integrin receptors CD11 b, CD18 and CD11 a, and systolic blood pressure, blood transfusion, lactate and base deficit as indices of shock. CD11 b expression on circulating neutrophils was increased 6 and 12 h after trauma. After correcting for the other shock indices, base deficit predicted CD11 b expression at 12 h. CD11 b expression was negatively correlated with the circulating neutrophil count. CONCLUSIONS: The degree of metabolic acidosis after trauma correlates directly with CD11 b receptor expression on circulating neutrophils. This relation may be the mechanism whereby post-traumatic shock results in neutrophil sequestration and neutrophil-mediated organ injury and failure.
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Acidosis/complicaciones , Antígeno de Macrófago-1/metabolismo , Insuficiencia Multiorgánica/etiología , Neutrófilos/fisiología , Choque Traumático/metabolismo , Adolescente , Adulto , Análisis de Varianza , Antígenos CD18/metabolismo , Humanos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/efectos adversos , Persona de Mediana Edad , Estudios Prospectivos , Análisis de Regresión , Choque Traumático/complicaciones , Choque Traumático/fisiopatología , Factores de Tiempo , Regulación hacia Arriba/fisiologíaRESUMEN
Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.
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Regulación de la Expresión Génica/efectos de los fármacos , Receptores de Ácido Retinoico/metabolismo , Retinoides/farmacología , Factores de Transcripción/metabolismo , Animales , Blastocisto , ADN/metabolismo , Embrión no Mamífero/efectos de los fármacos , Células Madre de Carcinoma Embrionario , Humanos , Ligandos , Ratones , Células Madre Neoplásicas , Fragmentos de Péptidos , Regiones Promotoras Genéticas/genética , Conformación Proteica/efectos de los fármacos , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes de Fusión , Receptores X Retinoide , Teratógenos/farmacología , Factores de Transcripción/química , Activación Transcripcional , Xenopus/embriología , Pez Cebra/embriologíaRESUMEN
Structurally diverse peroxisome proliferators and related compounds that have been demonstrated to induce the ligand-dependent transcriptional activation function of mouse peroxisome proliferator-activated receptor alpha (mPPARalpha) in transfection experiments were tested for the ability to induce conformational changes within mPPARalpha in vitro. WY-14,643, 5,8,11,14-eicosatetraynoic acid, LY-171883, and clofibric acid all directly induced mPPARalpha conformational changes as evidenced by a differential protease sensitivity assay. Carboxyl-terminal truncation mutagenesis of mPPARalpha differentially affected the ability of these ligands to induce conformational changes suggesting that PPAR ligands may make distinct contacts with the receptor. Direct interaction of peroxisome proliferators and related compounds with, and the resulting conformational alteration(s) in, mPPARalpha may facilitate interaction of the receptor with transcriptional intermediary factors and/or the general transcription machinery and, thus, may underlie the molecular basis of ligand-dependent transcriptional activation mediated by mPPARalpha.
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Receptores Citoplasmáticos y Nucleares/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Quimotripsina/metabolismo , Hidrólisis , Ligandos , Ratones , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor alpha in a ligand-enhanced manner. The PPARalpha-interacting domain of p300 was mapped to amino acids 39-117 which interacted strongly with PPARalpha but did not interact with retinoic acid receptor-gamma or retinoid X receptor-alpha. Amino acids within the carboxyl terminus of PPARalpha as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARalpha and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARalpha and further characterize the molecular mechanisms of PPARalpha-mediated transcriptional regulation.