ABSTRACT
En el marco de preparación de un Plan nacional hacia el envejecimiento saludable, la representación de la OPS/OMS en Bolivia, en coordinación con la Unidad de redes área del continuo de atención del Ministerio de Salud, decidió profundizar el conocimiento sobre la practica del autocuidado en personas adultas mayores y su nexo con los servicios de atención primaria. El estudio exploratorio concluyo un encuesta transversal cuantitativa a una muestra de personas mayores de 4 municipios del departamento de La Paz y grupos focales complementarios. En paralelo se hizo una observación sistemática del desempeño de 7 centros de salud de las mismas zonas de estudio, con entrevistas a personal clave y documentación visual. Los hallazgos de la observación del desempeño de los centros de salud confirman la hipótesis que la promoción de la salud en general y en particular para el autocuidado y para las personas mayores quedaron relegados desde la planificación de las actividades, la definición de y reporte sobre indicadores la disponibilidad de recursos humanos y la inversión en materiales educativos, a pesar de ser parte de la política SAFCI. Los centros de atención son la fuente de información de menos de 20% de las personas entrevistadas y fuentes no confiables como la radio y la televisión, seguidos de los pares, son declarados como fuentes de información en salud, con diferencias significativas por nivel de educación y lugar de vida no por género o grupos de edad. No solo es necesario invertir de una vez recursos para la promoción y el autocuidado a partir de los centros de salud, sino también adaptar la metodología de información y los contenidos para una audiencia heterogénea, pero e su mayoría con altos niveles de analfabetismo en salud.
Subject(s)
Self CareABSTRACT
Mammary gland homeostasis is regulated by both endogenous and exogenous signals, creating a balance between proliferation and apoptosis. It is thought that breast cancer develops from the acquisition of multiple genetic changes. The function of tumor suppressor p53 is fequently lost in cancers; however, not all cells that lose p53 progress to become invasive cancer. We have developed a model of early mammary carcinogenesis to investigate some of the internal and external signaling pathways that target the elimination ot normal basal-type human mammary epithelial cells (HMECs) that acutely acquire p53-damage. Here, we show that both tamoxifen (Tam) and three-dimensional prepared extracellular matrix culture (3-D rECM) induce apoptosis in HMEC cells with acute loss of p53 [*p53(-) HMECs] through induction of interferon regulatory factor-1 (IRF-1). Tam and rECM signaling in *p53(-) HMECs (1) promotes the recruitment of a STAT1/ CBP complex to the IRF-1 promoter, (2) upregulates IRF-1, (3) activates caspase-1 and -3, and (4) induces apoptosis. Suppression of IRF-1 with siRNA oligos inhibited both Tam- and rECM-induced apoptosis. These observations demonstrate that IRF-1 plays a critical role in eliminating p53-damaged cells, and may play a more global role in mammary gland homeostasis.
Subject(s)
Apoptosis/physiology , Breast Neoplasms/metabolism , Breast/pathology , Cell Transformation, Neoplastic , Interferon Regulatory Factor-1/physiology , Models, Biological , Tumor Suppressor Protein p53/metabolism , Breast/metabolism , Breast Neoplasms/pathology , Caspases/metabolism , Cell Culture Techniques , Cells, Cultured , Extracellular Matrix/metabolism , Humans , STAT1 Transcription Factor/metabolism , Tamoxifen/pharmacology , Tumor Suppressor Protein p53/geneticsABSTRACT
PURPOSE: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. EXPERIMENTAL DESIGN: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. RESULTS: INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-beta2, estrogen receptor-alpha, and breast cancer-associated 1 genes (P = 0.001). CONCLUSIONS: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.
Subject(s)
Breast Neoplasms/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA Methylation , Gene Silencing , Mammary Glands, Human/metabolism , Adult , Aged , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Humans , Mammary Glands, Human/cytology , Middle Aged , Promoter Regions, Genetic , RiskABSTRACT
Interactions between normal mammary epithelial cells and extracellular matrix (ECM) are important for mammary gland homeostasis. Loss of interactions between ECM and normal mammary epithelial cells are thought to be an early event in mammary carcinogenesis. CREB-binding protein (CBP) is an important regulator of proliferation and apoptosis but the role of CBP in ECM signaling is poorly characterized. CBP was suppressed in basal-cytokeratin-positive HMECs (CK5/6+, CK14+, CK8-, CK18-, CK19-). Suppression of CBP resulted in loss of reconstituted ECM-mediated growth control and apoptosis and loss of laminin-5 alpha3-chain expression. Suppression of CBP in normal human mammary epithelial cells (HMECs) resulted in loss of CBP occupancy of the LAMA3A promoter and decreased LAMA3A promoter activity and laminin-5 alpha-3 chain expression. Exogenous expression of CBP in CBP-negative HMECs that have lost reconstituted ECM-mediated growth regulation and apoptosis resulted in (1) CBP occupancy of the LAMA3A promoter, (2) increased LAMA3A activity and laminin-5 alpha3-chain expression, and (3) enhancement of reconstituted ECM-mediated growth regulation and apoptosis. Similarly, suppression of laminin-5 alpha3-chain expression in HMECs resulted in loss of reconstituted ECM-mediated growth control and apoptosis. These observations suggest that loss of CBP in basal-cytokeratin-positive HMECs results in loss of reconstituted ECM-mediated growth control and apoptosis through loss of LAMA3A activity and laminin-5 alpha3-chain expression. Results in these studies may provide insight into early events in basal-type mammary carcinogenesis.
Subject(s)
Apoptosis/physiology , CREB-Binding Protein/physiology , Cell Proliferation , Epithelial Cells/cytology , Extracellular Matrix/metabolism , Laminin/physiology , Mammary Glands, Human/cytology , Mammary Glands, Human/physiology , Apoptosis/genetics , CREB-Binding Protein/antagonists & inhibitors , CREB-Binding Protein/genetics , CREB-Binding Protein/metabolism , Cell Polarity/genetics , Cells, Cultured , Chromosomes, Human, Pair 16/genetics , Chromosomes, Human, Pair 16/metabolism , Down-Regulation/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Extracellular Matrix/pathology , Extracellular Matrix/physiology , Gene Rearrangement/genetics , Humans , Laminin/antagonists & inhibitors , Laminin/biosynthesis , Laminin/genetics , Mammary Glands, Human/pathology , Promoter Regions, Genetic , Protein Binding/genetics , Up-Regulation/geneticsABSTRACT
The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.
Subject(s)
Plasminogen Activator Inhibitor 1/physiology , Protease Inhibitors/pharmacology , Alanine/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cattle , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Kinetics , Models, Chemical , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/metabolism , Serpins/metabolism , Spectrometry, Fluorescence , Spectrophotometry , Swine , Thermodynamics , Trypsin/pharmacologyABSTRACT
In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.
Subject(s)
Enzyme Inhibitors/pharmacology , Glutathione Transferase/metabolism , Prodrugs/metabolism , Antineoplastic Agents/pharmacology , Binding, Competitive , Catalysis , Chromatography, Liquid , Enzyme Activation , Esterases/metabolism , Esters/metabolism , Ethacrynic Acid/metabolism , Glutathione/metabolism , Glutathione S-Transferase pi , Glutathione Transferase/chemistry , Humans , Hydrolysis , Isoenzymes/chemistry , Kinetics , Methyltransferases , Models, Chemical , Spectrometry, Mass, Electrospray Ionization , Sulfhydryl Compounds/metabolism , Time FactorsABSTRACT
El proyecto anticipo la implementacion del Plan Nacional del Adulto Mayor, del Plan Internacional del Envejecimiento de Madrid de 2002, y del proximo Plan Regional a firmarse en noviembre de 2003, planes que fomentan la participacion de todos los actores, autoridades y comunidad. Interviene en un contexto de desarrollo de las leyes de Participacion Popular, Municipal y Descentralizacion, en el contexto de la Estrategia de Reduccion de la Pobreza. En este contexto, el proyecto se convierte en el experimento de una estrategia de intervencion, la cual revela ser una experiencia de buena practica que hay que difundir, integrar y repetir, tal cual o con modificaciones. Se realizaron estudios de caso en Sucre, Coroico, La Paz y EL Alto.
Subject(s)
Healthy City , Aging , National Planning , Bolivia , Project Formulation , Participatory PlanningABSTRACT
El proyecto anticipo la implementacion del Plan Nacional del Adulto Mayor, del Plan Internacional del Envejecimiento de Madrid de 2002, y del proximo Plan Regional a firmarse en noviembre de 2003, planes que fomentan la participacion de todos los actores, autoridades y comunidad. Interviene en un contexto de desarrollo de las leyes de Participacion Popular, Municipal y Descentralizacion, en el contexto de la Estrategia de Reduccion de la Pobreza. En este contexto, el proyecto se convierte en el experimento de una estrategia de intervencion, la cual revela ser una experiencia de buena practica que hay que difundir, integrar y repetir, tal cual o con modificaciones. Se realizaron estudios de caso en Sucre, Coroico, La Paz y EL Alto.
Subject(s)
Healthy City , Aging , National Planning , Bolivia , Project Formulation , Participatory PlanningABSTRACT
The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.
Subject(s)
Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Glutathione/analogs & derivatives , Tyrosine/chemistry , Binding Sites , Catalysis , Chemical Phenomena , Chemistry, Physical , Fluorescence Polarization , Gene Expression , Glutathione/metabolism , Glutathione Transferase/genetics , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
Reunir toda la información posible sobre la situación del adulto mayor dentro del municipio, utilizando los resultatos y la información recolectados para poder determinar las intervenciones a corto y mediano plazo, y a la vez para servir la linea de base para evaluacion de impacto de los proyectos futuros.(au)
Subject(s)
Humans , Male , Adult , Female , Aged , Adult Health , Adult , Healthy City , BoliviaABSTRACT
Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathione binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Glutathione/analogs & derivatives , Glutathione/chemistry , Glutathione/metabolism , Models, Molecular , Apoenzymes/chemistry , Apoenzymes/metabolism , Binding Sites , Crystallography, X-Ray , Glycerol/chemistry , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Isoenzymes , Ligands , Protein Conformation , Sensitivity and Specificity , Tyrosine/chemistry , Water/chemistryABSTRACT
Se propone investigar las condiciones de salud y el contexto social general de las personas mayores de 60 años dentro del municipio a traves de una encuesta transversal, principalmente descriptiva, en la cual se recolectan datos cualitativos y cuantitativos sobre la salud y la situación social de las personas mayores.(au)
