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The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.
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Factor de Transcripción GATA3/metabolismo , Transducción de Señal , Linfocitos T , Animales , Diferenciación Celular , Linaje de la Célula , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Redes Reguladoras de Genes , Ratones , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.
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Ensamble y Desensamble de Cromatina , Suturas Craneales/crecimiento & desarrollo , Craneosinostosis/metabolismo , Mutación Missense , Nucleosomas/metabolismo , Osteogénesis , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Animales , Suturas Craneales/metabolismo , Craneosinostosis/genética , Craneosinostosis/fisiopatología , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Humanos , Lactante , Masculino , Ratones , Unión Proteica , Conformación Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Proteína 4 de Unión a Retinoblastoma/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Población Blanca , Secuenciación Completa del GenomaRESUMEN
OBJECTIVES: Promoting patient involvement in managing co-occurring physical and mental health conditions is increasingly recognized as critical to improving outcomes and controlling costs in this growing chronically ill population. The main objective of this study was to conduct an economic evaluation of the Wellness Incentives and Navigation (WIN) intervention as part of a longitudinal randomized pragmatic clinical trial for chronically ill Texas Medicaid enrollees with co-occurring physical and mental health conditions. METHODS: The WIN intervention used a personal navigator, motivational interviewing, and a flexible wellness expense account to increase patient activation, that is, the patient's knowledge, skills, and confidence in managing their self-care and co-occurring physical and mental health conditions. Regression models were fit to both participant-level quality-adjusted life years (QALYs) and total costs of care (including the intervention) controlling for demographics, health status, poverty, Medicaid managed care plan, intervention group, and baseline health utility and costs. Incremental costs and QALYs were calculated based on the difference in predicted costs and QALYs under intervention versus usual care and were used to calculate the incremental cost-effectiveness ratios (ICERs). Confidence intervals were calculated using Fieller's method, and sensitivity analyses were performed. RESULTS: The mean ICER for the intervention compared with usual care was $12 511 (95% CI $8971-$16 842), with a sizable majority of participants (70%) having ICERs below $40 000. The WIN intervention also produced higher QALY increases for participants who were sicker at baseline compared to those who were healthier at baseline. CONCLUSION: The WIN intervention shows considerable promise as a cost-effective intervention in this challenging chronically ill population.
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Conocimientos, Actitudes y Práctica en Salud , Promoción de la Salud/organización & administración , Medicaid/estadística & datos numéricos , Afecciones Crónicas Múltiples/epidemiología , Adulto , Análisis Costo-Beneficio , Femenino , Promoción de la Salud/economía , Estado de Salud , Humanos , Estudios Longitudinales , Masculino , Medicaid/economía , Entrevista Motivacional/organización & administración , Navegación de Pacientes/organización & administración , Años de Vida Ajustados por Calidad de Vida , Autocuidado , Autoimagen , Factores Socioeconómicos , Texas/epidemiología , Estados Unidos , Adulto JovenRESUMEN
Ewing's sarcoma (EwS) is the second most common bone cancer in children and adolescents. Current chemotherapy regimens are mainly ineffective in patients with relapsed disease and cause long-term effects in survivors. Therefore, we have developed a combinatorial therapy based on a novel drug candidate named ML111 that exhibits selective activity against EwS cells and synergizes with vincristine. To increase the aqueous solubility of hydrophobic ML111, polymeric nanoparticles (ML111-NP) were developed. In vitro data revealed that ML111-NP compromise viability of EwS cells without affecting non-malignant cells. Furthermore, ML111-NP exhibit strong synergistic effects in a combination with vincristine on EwS cells, while this drug pair exhibits antagonistic effects towards normal cells. Finally, animal studies validated that ML111-NP efficiently accumulate in orthotopic EwS xenografts after intravenous injection and provide superior therapeutic outcomes in a combination with vincristine without evident toxicity. These results support the potential of the ML111-based combinatorial therapy for EwS.
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Antineoplásicos , Sinergismo Farmacológico , Sarcoma de Ewing , Vincristina , Animales , Humanos , Ratones , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Nanopartículas/química , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Vincristina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylation-dephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five residues apart.
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Espectrometría de Masas/métodos , Proteínas Represoras/metabolismo , Timocitos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcimicina/farmacología , Ionóforos de Calcio/farmacología , Células Cultivadas , Immunoblotting , Cinética , Lisina/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Forbol 12,13-Dibutirato/farmacología , Fosforilación/efectos de los fármacos , Serina/metabolismo , Sumoilación/efectos de los fármacos , Treonina/metabolismo , Timocitos/citología , Factores de TiempoRESUMEN
The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.
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Sistema de Señalización de MAP Quinasas , Proteínas Represoras/metabolismo , Sumoilación , Timo/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Calcimicina/farmacología , Línea Celular , Células Cultivadas , Humanos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteínas Represoras/química , Homología de Secuencia de Aminoácido , Timo/citología , Proteínas Supresoras de Tumor/químicaRESUMEN
Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.
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Diferenciación Celular , Células Precursoras de Linfocitos T/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular/inmunología , Linaje de la Célula , Células Cultivadas , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos T/citología , Unión Proteica , Elementos Reguladores de la Transcripción/inmunología , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Timo/citología , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Activación Transcripcional/inmunología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Pt-Ru binary catalysts were prepared on a polyaniline-functionalized multiwalled carbon nanotube (PANi/MWCNT). PANi/MWCNT composites were synthesized by the polymerization of aniline in the presence of a carbon nanotube suspension using FeSO(4) and (NH(4))(2)S(2)O(8) as the oxidants. The Pt-Ru/PANi/MWCNT catalysts were formed by the chemical reduction of H(2)PtCl(6) and RuCl(3) using NaBH(4) as the reducing agent. The binary component catalyst is sharply distributed, with particle sizes ranging from 2.0 to 4.0 nm, and the Pt and Ru distributions are homogeneous when supported on PANi/MWCNT. In comparison, the binary catalyst supported on bare MWCNT displayed a Pt-rich core and a Ru-rich shell nanostructure. The surface composition deduced from CO stripping potentials confirms that the Ru surface content (χ(Ru)) is approximately 50% for the Pt-Ru alloy on PANi/MWCNT, and the catalyst on bare MWCNT shows nearly 70% Ru on the surface. Pt-Ru binary catalysts supported on PANi/MWCNT have higher activity, a higher Pt utilization efficiency, and much better durability when compared to other catalyst supports on bare MWCNT or on Vulcan XC-72.
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Compuestos de Anilina/química , Nanoestructuras/química , Nanotubos de Carbono/química , Platino (Metal)/química , Rutenio/química , Catálisis , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Ewing's sarcoma, characterized by pathognomonic t (11; 22) (q24; q12) and related chromosomal ETS family translocations, is a rare aggressive cancer of bone and soft tissue. Current protocols that include cytotoxic chemotherapeutic agents effectively treat localized disease; however, these aggressive therapies may result in treatment-related morbidities including second-site cancers in survivors. Moreover, the five-year survival rate in patients with relapsed, recurrent, or metastatic disease is less than 30%, despite intensive therapy with these cytotoxic agents. By using high-throughput phenotypic screening of small molecule libraries, we identified a previously uncharacterized compound (ML111) that inhibited in vitro proliferation of six established Ewing's sarcoma cell lines with nanomolar potency. Proteomic studies show that ML111 treatment induced prometaphase arrest followed by rapid caspase-dependent apoptotic cell death in Ewing's sarcoma cell lines. ML111, delivered via methoxypoly(ethylene glycol)-polycaprolactone copolymer nanoparticles, induced dose-dependent inhibition of Ewing's sarcoma tumor growth in a murine xenograft model and invoked prometaphase arrest in vivo, consistent with in vitro data. These results suggest that ML111 represents a promising new drug lead for further preclinical studies and is a potential clinical development for the treatment of Ewing's sarcoma.
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Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.
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Depsipéptidos/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Canales de Translocación SEC/efectos de los fármacos , Sitios de Unión , Células Cultivadas , Depsipéptidos/metabolismo , Humanos , Proteínas de la Membrana/biosíntesis , Etiquetas de Fotoafinidad/química , Canales de Translocación SEC/metabolismoRESUMEN
Phospholipase C-beta (PLC-beta) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-beta1 and PLC-beta3 bound immobilized PIP(3). In this study, PIP(3) was found to potentiate Ca(2+)-stimulated PLC-beta activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP(3) accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP(3) accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP(3) in directly potentiating PLC-beta activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-beta3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-beta is not important for p110CAAX-induced membrane association.
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Isoenzimas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfolipasa C beta/metabolismo , Transducción de Señal/fisiología , Androstadienos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Cromonas/metabolismo , Inhibidores Enzimáticos/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Morfolinas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfolipasa C beta/antagonistas & inhibidores , Fosfolipasa C beta/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , WortmaninaRESUMEN
OBJECTIVE: To examine whether the Wellness Incentive and Navigation (WIN) intervention can improve health-related quality of life (HRQOL) among Medicaid enrollees with co-occurring physical and behavioral health conditions. DATA SOURCES: Annual telephone survey data from 2013 to 2016, linked with claims data. STUDY DESIGN: We recruited 1259 participants from the Texas STAR + PLUS managed care program and randomized them into an intervention group that received flexible wellness accounts and navigator services or a control group that received standard care. We conducted 4 waves of telephone surveys to collect data on HRQOL, patient activation, and other participant demographic and clinical characteristics. DATA COLLECTION/EXTRACTION METHODS: The 3M Clinical Risk Grouping Software was used to extract variables from claims data and group participants based on disease severity. PRINCIPAL FINDINGS: Our results showed that the WIN intervention was effective in increasing patient activation and HRQOL among Medicaid enrollees with co-occurring physical and behavioral health conditions. Furthermore, we found that this intervention effect on HRQOL was partially mediated by patient activation. CONCLUSIONS: Providing navigator support with wellness account is effective in improving HRQOL among Medicaid enrollees. The pragmatic nature of the trial maximizes the chance of successfully implementing it in state Medicaid programs.
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Conductas Relacionadas con la Salud , Promoción de la Salud/métodos , Medicaid/estadística & datos numéricos , Motivación , Navegación de Pacientes/métodos , Participación del Paciente/psicología , Calidad de Vida/psicología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Participación del Paciente/estadística & datos numéricos , Encuestas y Cuestionarios , Texas , Estados UnidosRESUMEN
A recent genome-wide association study found that genetic variants on chromosomes 3, 6, 7, 10, 11, 19 and X were associated with prostate cancer risk. We evaluated the most significant single-nucleotide polymorphisms (SNP) in these loci using a worldwide consortium of 13 groups (PRACTICAL). Blood DNA from 7,370 prostate cancer cases and 5,742 male controls was analyzed by genotyping assays. Odds ratios (OR) associated with each genotype were estimated using unconditional logistic regression. Six of the seven SNPs showed clear evidence of association with prostate cancer (P = 0.0007-P = 10(-17)). For each of these six SNPs, the estimated per-allele OR was similar to those previously reported and ranged from 1.12 to 1.29. One SNP on 3p12 (rs2660753) showed a weaker association than previously reported [per-allele OR, 1.08 (95% confidence interval, 1.00-1.16; P = 0.06) versus 1.18 (95% confidence interval, 1.06-1.31)]. The combined risks associated with each pair of SNPs were consistent with a multiplicative risk model. Under this model, and in combination with previously reported SNPs on 8q and 17q, these loci explain 16% of the familial risk of the disease, and men in the top 10% of the risk distribution have a 2.1-fold increased risk relative to general population rates. This study provides strong confirmation of these susceptibility loci in multiple populations and shows that they make an important contribution to prostate cancer risk prediction.
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Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Lesiones Precancerosas/genética , Próstata/patología , Neoplasias de la Próstata/genética , Alelos , Estudios de Casos y Controles , Variación Genética , Genotipo , Humanos , Modelos Logísticos , Masculino , Lesiones Precancerosas/patología , Neoplasias de la Próstata/patología , RiesgoRESUMEN
Phospholipase C-beta (PLC-beta) isozymes (EC 3.1.4.11) hydrolyze the membrane phospholipid phosphatidylinositol-4,5-bisphosphate to generate intracellular second messenger signaling molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation and other cellular stimuli. PLCbeta1 and PLCbeta3 isozymes were previously demonstrated to bind the calcium-sensitive molecule calmodulin [McCullar JS, Larsen SA, Millimaki RA, Filtz TM. Calmodulin is a phospholipase C-{beta} interacting protein. J Biol Chem 2003;278(36):33708-13]. We have now shown through fluorescence anisotropy that calmodulin/PLCbeta3 affinities increase with increasing calcium in a physiologically relevant concentration range. The bimolecular affinity constants for calmodulin interaction with PLCbeta1 or PLCbeta3 were estimated as 260 and 200 nM, respectively, from fluorescence anisotropy data. There was no effect of calmodulin on basal or G alpha q-stimulated catalytic activity for either isozyme. However, the interaction between calmodulin and PLCbeta3 leads to potentiation of activation by the G-protein beta gamma dimer in an in vitro assay. 1321N1 cells treated with calmodulin inhibitors concurrent with and post-stimulation of muscarinic receptors significantly reduced [3H]PIP hydrolysis. Together these data are suggestive of cooperative role for calmodulin in the G-protein beta gamma dimer-stimulated activity of PLCbeta3.
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Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Isoenzimas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Línea Celular , Activación Enzimática , Polarización de Fluorescencia , Hidrólisis , Fosfatidilinositoles/metabolismo , Fosfolipasa C beta , Receptores Muscarínicos/metabolismoRESUMEN
Myosins are actin-based molecular motors that may have specialized trafficking and contractile functions in cytoskeletal compartments that lack microtubules. The postsynaptic excitatory synapse is one such specialization, yet little is known about the spatial organization of myosin motor proteins in the mature brain. We used a proteomics approach to determine if class II and class V myosin isoforms are associated with Triton X-100-resistant membranes isolated from mouse forebrain. Two nonmuscle myosin isoforms (II-B and Va), were identified as components of lipid raft fractions that also contained typical membrane skeletal proteins such as non-erythrocyte spectrins, actin, alpha-actinin-2 and tubulin subunits. Other raft-associated proteins included lipid raft markers, proteins involved in cell adhesion and membrane dynamics, receptors and channels including glutamate receptor subunits, scaffolding and regulatory proteins. Myosin II-B and Va were also present in standard postsynaptic density (PSD) fractions, however retention of myosin II-B was strongly influenced by ATP status. If homogenates were supplemented with ATP, myosin II-B could be extracted from PSD I whereas myosin Va and other postsynaptic proteins were resistant to extraction. In summary, both myosin isoforms are components of a raft-associated membrane skeleton and are likely detected in standard PSD fractions as a result of their intrinsic ability to form actomyosin. Myosin II-B, however, is more loosely associated with PSD fractions than myosin Va, which appears to be a core PSD protein.
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Membrana Celular/ultraestructura , Citoesqueleto/efectos de los fármacos , Detergentes/farmacología , Miosina Tipo V/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Prosencéfalo/ultraestructura , Adenosina Trifosfato/farmacología , Animales , Fraccionamiento Celular/métodos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Espectrometría de Masas/métodos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Ratones , Neuronas/ultraestructura , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Transcription factors comprise just over 7% of the human proteome and serve as gatekeepers of cellular function, integrating external signal information into gene expression programs that reconfigure cellular physiology at the most basic levels. Surface-initiated cell signaling pathways converge on transcription factors, decorating these proteins with an array of post-translational modifications (PTMs) that are often interdependent, being linked in time, space, and combinatorial function. These PTMs orchestrate every activity of a transcription factor over its entire lifespan--from subcellular localization to protein-protein interactions, sequence-specific DNA binding, transcriptional regulatory activity, and protein stability--and play key roles in the epigenetic regulation of gene expression. The multitude of PTMs of transcription factors also offers numerous potential points of intervention for development of therapeutic agents to treat a wide spectrum of diseases. We review PTMs most commonly targeting transcription factors, focusing on recent reports of sequential and linked PTMs of individual factors.
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Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , HumanosRESUMEN
BACKGROUND: Cell types are defined at the molecular level during embryogenesis by a process called pattern formation and created by the selective utilization of combinations of sequence specific transcription factors. Developmental programs define the sets of genes that are available to each particular cell type, and real-time biochemical signaling interactions define the extent to which these sets are used at any given time and place. Gene expression is regulated through the integrated action of many cis-regulatory elements, including core promoters, enhancers, silencers, and insulators. The chromatin state in developing body parts provides a code to cellular populations that direct their cell fates. Chromatin profiling has been a method of choice for mapping regulatory sequences in cells that go through developmental transitions. RESULTS: We used antibodies against histone H3 lysine 4 trimethylations (H3K4me3) a modification associated with promoters and open/active chromatin, histone H3 lysine 27 trimethylations (H3K27me3) associated with Polycomb-repressed regions and RNA polymerase II (Pol2) associated with transcriptional initiation to identify the chromatin state signature of the mouse forelimb during mid-gestation, at embryonic day 12 (E12). The families of genes marked included those related to transcriptional regulation and embryogenesis. One third of the marked genes were transcriptionally active while only a small fraction were bivalent marked. Sequence specific transcription factors that were activated were involved in cell specification including bone and muscle formation. CONCLUSION: Our results demonstrate that embryonic limb cells do not exhibit the plasticity of the ES cells but are rather programmed for a finer tuning for cell lineage specification.
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The synthesis of a modular colloidal polymer system based on the dipolar assembly of CdSe@CdS nanorods functionalized with a single cobalt nanoparticle "tip" (CoNP-tip) is reported. These heterostructured nanorods spontaneously self-assembled via magnetic dipolar associations of the cobalt domains. In these assemblies, CdSe@CdS nanorods were carried as densely grafted side chain groups along the dipolar NP chain to form bottlebrush-type colloidal polymers. Nanorod side chains strongly affected the conformation of individual colloidal polymer bottlebrush chains and the morphology of thin films. Dipolar CoNP-tipped nanorods were then used as "colloidal monomers" to form mesoscopic assemblies reminiscent of traditional copolymers possessing segmented and statistical compositions. Investigation of the phase behavior of colloidal polymer blends revealed the formation of mesoscopic phase separated morphologies from segmented colloidal copolymers. These studies demonstrated the ability to control colloidal polymer composition and morphology in a manner observed for classical polymer systems by synthetic control of heterostructured nanorod structure and harnessing interparticle dipolar associations.
RESUMEN
A methodology providing access to dumbbell-tipped, metal-semiconductor and metal oxide-semiconductor heterostructured nanorods has been developed. The synthesis and characterization of CdSe@CdS nanorods incorporating ferromagnetic cobalt nanoinclusions at both nanorod termini (i.e., dumbbell morphology) are presented. The key step in the synthesis of these heterostructured nanorods was the decoration of CdSe@CdS nanorods with platinum nanoparticle tips, which promoted the deposition of metallic CoNPs onto Pt-tipped CdSe@CdS nanorods. Cobalt nanoparticle tips were then selectively oxidized to afford CdSe@CdS nanorods with cobalt oxide domains at both termini. In the case of longer cobalt-tipped nanorods, heterostructured nanorods were observed to self-organize into complex dipolar assemblies, which formed as a consequence of magnetic associations of terminal CoNP tips. Colloidal polymerization of these cobalt-tipped nanorods afforded fused nanorod assemblies from the oxidation of cobalt nanoparticle tips at the ends of nanorods via the nanoscale Kirkendall effect. Wurtzite CdS nanorods survived both the deposition of metallic CoNP tips and conversion into cobalt oxide phases, as confirmed by both XRD and HRTEM analysis. A series of CdSe@CdS nanorods of four different lengths ranging from 40 to 174 nm and comparable diameters (6-7 nm) were prepared and modified with both cobalt and cobalt oxide tips. The total synthesis of these heterostructured nanorods required five steps from commercially available reagents. Key synthetic considerations are discussed, with particular emphasis on reporting isolated yields of all intermediates and products from scale up of intermediate precursors.
Asunto(s)
Compuestos de Cadmio/química , Cobalto/química , Nanotubos/química , Nanotubos/ultraestructura , Platino (Metal)/química , Compuestos de Selenio/química , Sulfuros/química , Cristalización/métodos , Sustancias Macromoleculares/química , Campos Magnéticos , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Carbon supported Au-Pt catalysts with different bimetallic compositions were prepared by water-in-oil (w/o) micro-emulsions. Carbon Vulcan XC-72R was added during the synthesis of particles in order to obtain good dispersion. Structural characterization was performed using XRD (X-ray diffraction) at wide angles, WAXS (wide-angle X-ray scattering) which showed that Pt-Au particles exhibited alloy properties in samples with high gold-content, and a segregation effect for those with low gold-content. Electrochemical characterization allowed estimation of the surface composition of Pt-Au alloys. These experiments have been confirmed by XRD data. Moreover, HRTEM (high resolution transmission electron microscopy) and XEDS (X-ray energy dispersive spectroscopy) characterization further confirmed the electrochemical results and XRD data. CO stripping experiments have shown an increasing bonding energy between CO and platinum with the gold content in the nanoalloys.