Asunto(s)
Infección Hospitalaria , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Antibacterianos/uso terapéutico , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Atención a la Salud , Humanos , Meticilina , Resistencia a la Meticilina , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/prevención & controlRESUMEN
BACKGROUND: Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years, the molecular mechanism accounting for this activity is still unclear. OBJECTIVES AND METHODS: To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions, using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. RESULTS: We identified 328 annotated genes differentially regulated by anagrelide, including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3, whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4, a known TRIB3 inducer. Notably, immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α, which is an upstream regulator of ATF4, and increased ATF4 protein levels. Furthermore, salubrinal, an inhibitor of eIF2α dephosphorylation, increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. CONCLUSIONS: These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis.
Asunto(s)
Perfilación de la Expresión Génica , Fármacos Hematológicos/farmacología , Megacariocitos/efectos de los fármacos , Quinazolinas/farmacología , Trombopoyesis/efectos de los fármacos , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Biología Computacional , Bases de Datos Genéticas , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Megacariocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Trombopoyesis/genética , Factores de TiempoRESUMEN
Peroxisome proliferator-activated receptor gamma (PPARγ) is known to be activated via exercise-associated transient increases in oxidative stress. However, the precise mechanism(s) triggering PPARγ activation in monocytes during/following exercise remain to be confirmed. Here, two cohorts of five healthy male individuals undertook exercise bouts (cycling; 70% VO2max; 45 min) in the presence/absence of dietary antioxidant supplementation (vitamins C (1000 mg/day) and E (400IU/day) for four weeks before exercise); monocytic 5' adenosine monophosphate-activated protein kinase (AMPK)/PPARγ co-activator-1alpha (PGC-1α)/PPARγ signalling was investigated in samples obtained before exercise and up to 24 h after exercise, while THP-1 cells were cultured as an in vitro monocyte model. In THP-1 cells, AMPKα1 was phosphorylated within 1h of menadione (15 µM)-triggered increases in [reactive oxygen species (ROS)]cyto, an effect which was followed by upregulation of PPARγ and several of its target genes (PGC-1α, liver X receptor alpha [LXRα] and ATP-binding cassette subfamily A, member 1 [ABCA1]; 24-72 h), with these effects being blunted by co-administration of vitamin C (62.5 µM). Conversely, treatment with oxidised low-density lipoprotein (oxLDL) (1 µg/mL; 24-72 h), but not non-oxidised LDL, upregulated the above PPARγ-regulated genes without affecting AMPKα1 phosphorylation. In vivo, dietary antioxidant supplementation (which is known to prevent exercise-triggered increases in oxLDL levels) blunted exercise-associated upregulation of the above PPARγ-regulated genes, but had no effect on exercise-associated transient [ROS]cyto increases, or on AMPK phosphorylation. These data suggest that exercise-associated PPARγ signalling effects appear, at least in monocytes, to be mediated by increased generation of PPARγ ligands via oxidation of lipoproteins (following exercise-associated transient increases in oxidative stress), rather than via [ROS]cyto-mediated AMPK activation. These findings may be of clinical relevance, as PPARγ activation in monocytes is associated with beneficial effects related to type-2 diabetes and its cardiovascular complications.
Asunto(s)
Proteínas Quinasas Activadas por AMP/sangre , Ejercicio Físico/fisiología , Lipoproteínas LDL/sangre , Monocitos/metabolismo , PPAR gamma/sangre , Adulto , Antioxidantes/administración & dosificación , Células Cultivadas , Estudios de Cohortes , Humanos , Lipoproteínas LDL/farmacología , Masculino , Estrés Oxidativo/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Transcripción/sangreRESUMEN
Chronic pain is a global public health problem, but the underlying molecular mechanisms are not fully understood. Here we examine genome-wide DNA methylation, first in 50 identical twins discordant for heat pain sensitivity and then in 50 further unrelated individuals. Whole-blood DNA methylation was characterized at 5.2 million loci by MeDIP sequencing and assessed longitudinally to identify differentially methylated regions associated with high or low pain sensitivity (pain DMRs). Nine meta-analysis pain DMRs show robust evidence for association (false discovery rate 5%) with the strongest signal in the pain gene TRPA1 (P=1.2 × 10(-13)). Several pain DMRs show longitudinal stability consistent with susceptibility effects, have similar methylation levels in the brain and altered expression in the skin. Our approach identifies epigenetic changes in both novel and established candidate genes that provide molecular insights into pain and may generalize to other complex traits.
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Canales de Calcio/genética , Metilación de ADN/genética , Hiperalgesia/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Canales de Potencial de Receptor Transitorio/genética , Gemelos Monocigóticos/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Epigénesis Genética , Femenino , Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Canal Catiónico TRPA1RESUMEN
The aim of the present study was to test the hypotheses that exercise is associated with generation of peroxisome proliferator-activated receptor-γ (PPARγ) ligands in the plasma and that this may activate PPARγ signaling within circulating monocytes, thus providing a mechanism to underpin the exercise-induced antiatherogenic benefits observed in previous studies. A cohort of healthy individuals undertook an 8-wk exercise-training program; samples were obtained before (Pre) and after (Post) standardized submaximal exercise bouts (45 min of cycling at 70% of maximal O(2) uptake, determined at baseline) at weeks 0, 4, and 8. Addition of plasma samples to PPARγ response element (PPRE)-luciferase reporter gene assays showed increased PPARγ activity following standardized exercise bouts (Post/Pre = 1.23 ± 0.10 at week 0, P < 0.05), suggesting that PPARγ ligands were generated during exercise. However, increases in PPARγ/PPRE-luciferase activity in response to the same standardized exercise bout were blunted during the training program (Post/Pre = 1.18 ± 0.14 and 1.10 ± 0.10 at weeks 4 and 8, respectively, P > 0.05 for both), suggesting that the relative intensity of the exercise may affect PPARγ ligand generation. In untrained individuals, specific transient increases in monocyte expression of PPARγ-regulated genes were observed within 1.5-3 h of exercise (1.7 ± 0.4, 2.6 ± 0.4, and 1.4 ± 0.1 fold for CD36, liver X receptor-α, and ATP-binding cassette subfamily A member 1, respectively, P < 0.05), with expression returning to basal levels within 24 h. In contrast, by the end of the exercise program, expression at the protein level of PPARγ target genes had undergone sustained increases that were not associated with an individual exercise bout (e.g., week 8 Pre/week 0 Pre = 2.79 ± 0.61 for CD36, P < 0.05). Exercise is known to upregulate PPARγ-controlled genes to induce beneficial effects in skeletal muscle (e.g., mitochondrial biogenesis and aerobic respiration). We suggest that parallel exercise-induced benefits may occur in monocytes, as monocyte PPARγ activation has been linked to beneficial antidiabetic effects (e.g., exercise-induced upregulation of monocytic PPARγ-controlled genes is associated with reverse cholesterol transport and anti-inflammatory effects). Thus, exercise-triggered monocyte PPARγ activation may constitute an additional rationale for prescribing exercise to type 2 diabetes patients.
Asunto(s)
Ejercicio Físico/fisiología , Metabolismo de los Lípidos/genética , PPAR gamma/genética , PPAR gamma/metabolismo , Transducción de Señal/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adulto , Antígenos CD36/genética , Antígenos CD36/metabolismo , Línea Celular Transformada , Estudios de Cohortes , Células HEK293 , Humanos , Inmunoglobulina A Secretora/genética , Inmunoglobulina A Secretora/metabolismo , Ligandos , Receptores X del Hígado , Monocitos/metabolismo , Monocitos/fisiología , Receptores Nucleares Huérfanos/metabolismo , Transducción de Señal/genética , Regulación hacia ArribaRESUMEN
OBJECTIVE: Pharmacological activation of the nuclear receptor PPARγ is linked to numerous beneficial effects in the contexts of inflammation, lipid homeostasis, Type-2 Diabetes (T2D) and atherosclerosis. These beneficial effects include priming of circulating monocytes for differentiation towards an 'alternative' anti-inflammatory M2 macrophage phenotype. As we have recently shown that participation in low-intensity exercise increases PPARγ expression and activity in leukocytes from previously sedentary individuals, we aimed to elucidate whether low-intensity exercise elicited a pattern of gene expression similar to that reported for M2 monocyte-macrophage differentiation. METHODS: 17 sedentary individuals undertook an 8-week low-intensity exercise programme (walking 10,000steps/day, three times/week). Changes in expression of PPARs and the PPARγ co-activators PGC-1α and PGC-1ß; Th2 (IL-4; IL-10) and Th1 (IL-6) cytokines; and markers for the M2 (AMAC1, CD14, MR, IL-4) and the 'classical' pro-inflammatory M1 (MCP-1, TNFα, IL-6) phenotypes, were determined using RT-PCR (to assess leukocyte mRNA expression) and ELISA (to assess plasma cytokine levels). RESULTS: Exercise was associated with upregulation of M2 markers, PGC-1α and PGC-1ß, and with downregulation of M1 markers. Moreover, plasma levels of Th2 cytokines increased after exercise, while those of Th1 cytokines decreased. However, other PPARs (PPARα; PPARß/δ) did not undergo marked exercise-induced activation or upregulation. Thus, participation in low-intensity exercise may prime monocytes for differentiation towards an M2 macrophage phenotype via PPARγ/PGC-1α/ß. CONCLUSION: Given the similarities between these effects and pharmacologically induced M2 polarisation, we propose that exercise-induced PPARγ/PGC-1α/ß-mediated M2 polarisation may constitute a novel anti-inflammatory benefit of low-intensity exercise.
Asunto(s)
Ejercicio Físico , Regulación de la Expresión Génica , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Leucocitos/metabolismo , PPAR gamma/metabolismo , Células Th2/citología , Adulto , Diferenciación Celular , Femenino , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
BACKGROUND: Anagrelide is a selective inhibitor of megakaryocytopoiesis used to treat thrombocytosis in patients with chronic myeloproliferative disorders. The effectiveness of anagrelide in lowering platelet counts is firmly established, but its primary mechanism of action remains elusive. OBJECTIVES AND METHODS: Here, we have evaluated whether anagrelide interferes with the major signal transduction cascades stimulated by thrombopoietin in the hematopoietic cell line UT-7/mpl and in cultured CD34(+) -derived human hematopoietic cells. In addition, we have used quantitative mRNA expression analysis to assess whether the drug affects the levels of known transcription factors that control megakaryocytopoiesis. RESULTS: In UT-7/mpl cells, anagrelide (1µm) did not interfere with MPL-mediated signaling as monitored by its lack of effect on JAK2 phosphorylation. Similarly, the drug did not affect the phosphorylation of STAT3, ERK1/2 or AKT in either UT-7/mpl cells or primary hematopoietic cells. In contrast, during thrombopoietin-induced megakaryocytic differentiation of normal hematopoietic cultures, anagrelide (0.3µm) reduced the rise in the mRNA levels of the transcription factors GATA-1 and FOG-1 as well as those of the downstream genes encoding FLI-1, NF-E2, glycoprotein IIb and MPL. However, the drug showed no effect on GATA-2 or RUNX-1 mRNA expression. Furthermore, anagrelide did not diminish the rise in GATA-1 and FOG-1 expression during erythropoietin-stimulated erythroid differentiation. Cilostamide, an exclusive and equipotent phosphodiesterase III (PDEIII) inhibitor, did not alter the expression of these genes. CONCLUSIONS: Anagrelide suppresses megakaryocytopoiesis by reducing the expression levels of GATA-1 and FOG-1 via a PDEIII-independent mechanism that is differentiation context-specific and does not involve inhibition of MPL-mediated early signal transduction events.
Asunto(s)
Factor de Transcripción GATA1/biosíntesis , Regulación de la Expresión Génica , Proteínas Nucleares/biosíntesis , Quinazolinas/farmacología , Receptores de Trombopoyetina/metabolismo , Factores de Transcripción/biosíntesis , Antígenos CD34/biosíntesis , Plaquetas/efectos de los fármacos , Línea Celular , Humanos , Megacariocitos/efectos de los fármacos , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Transducción de Señal , Transcripción GenéticaRESUMEN
General cognitive ability (g), which refers to what cognitive abilities have in common, is an important target for molecular genetic research because multivariate quantitative genetic analyses have shown that the same set of genes affects diverse cognitive abilities as well as learning disabilities. In this first autosomal genome-wide association scan of g, we used a two-stage quantitative trait locus (QTL) design with pooled DNA to screen more than 500,000 single nucleotide polymorphisms (SNPs) on microarrays, selecting from a sample of 7000 7-year-old children. In stage 1, we screened for allele frequency differences between groups pooled for low and high g. In stage 2, 47 SNPs nominated in stage 1 were tested by individually genotyping an independent sample of 3195 individuals, representative of the entire distribution of g scores in the full 7000 7-year-old children. Six SNPs yielded significant associations across the normal distribution of g, although only one SNP remained significant after a false discovery rate of 0.05 was imposed. However, none of these SNPs accounted for more than 0.4% of the variance of g, despite 95% power to detect associations of that size. It is likely that QTL effect sizes, even for highly heritable traits such as cognitive abilities and disabilities, are much smaller than previously assumed. Nonetheless, an aggregated 'SNP set' of the six SNPs correlated 0.11 (P < 0.00000003) with g. This shows that future SNP sets that will incorporate many more SNPs could be useful for predicting genetic risk and for investigating functional systems of effects from genes to brain to behavior.
Asunto(s)
Cognición/fisiología , ADN/genética , Genoma Humano/genética , Inteligencia/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Adolescente , Niño , ADN/análisis , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Genotipo , Humanos , Patrón de Herencia/genética , Estudios Longitudinales , Masculino , Herencia Multifactorial/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Carácter Cuantitativo HeredableRESUMEN
Active fluorescence (F) sensing systems have long been suggested as a means to identify species composition and determine physiological status of plants. Passive F systems for large-scale remote assessment of vegetation will undoubtedly rely on solar-induced F (SIF), and this information could potentially be obtained from the Fraunhofer line depth (FLD) principle. However, understanding the relationships between the information and knowledge gained from active and passive systems remains to be addressed. Here we present an approach in which actively induced F spectral data are used to simulate and project the magnitude of SIF that can be expected from near-ground observations within selected solar Fraunhofer line regions. Comparisons among vegetative species and nitrogen (N) supply treatments were made with three F approaches: the passive FLD principle applied to telluric oxygen (O2) bands from field-acquired canopy reflectance spectra, simulated SIF from actively induced laboratory emission spectra of leaves at a series of solar Fraunhofer lines ranging from 422 to 758 nm, and examination of two dual-F excitation algorithms developed from laboratory data. From these analyses we infer that SIF from whole-plant canopies can be simulated by use of laboratory data from active systems on individual leaves and that SIF has application for the large-scale assessment of vegetation.
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Agricultura/métodos , Algoritmos , Clorofila/análisis , Monitoreo del Ambiente/métodos , Oxígeno/análisis , Hojas de la Planta/química , Hojas de la Planta/crecimiento & desarrollo , Espectrometría de Fluorescencia/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We hypothesize that mild mental impairment (MMI) represents the low extreme of the same quantitative trait loci (QTLs) that operate throughout the distribution of intelligence. To detect QTLs of small effect size, we employed a direct association strategy by genotyping 432 presumably functional nonsynonymous single-nucleotide polymorphisms (nsSNPs) identified from public databases on DNA pools of 288 cases and 1025 controls. In total, 288 MMI cases were identified by in-home administration of McCarthy Scales of Children's Abilities to 836 twin pairs selected from a community sample of more than 14 000 children previously screened for nonverbal cognitive delay using parentally administered tests. Controls were selected from the community sample representing the full range of nonverbal intelligence. SNPs showing at least 7% allele frequency differences between case and control DNA pools were tested for their association with the full range of nonverbal intelligence using five DNA subpools, each representing quintiles of the normal quantitative trait scores from the 1025 controls. SNPs showing linear associations in the expected direction across quintiles using pooled DNA were individually genotyped for the 288 cases and 1025 controls and analyzed using standard statistical methods. One SNP (rs1136141) in HSPA8 met these criteria, yielding a significant (P=0.036) allelic frequency difference between cases and controls for individual genotyping and a significant (P=0.013) correlation within the control group that accounts for 0.5% of the variance. The present SNP strategy combined with DNA pooling and large samples represents a step towards identifying QTLs of small effect size associated with complex traits in the postgenomic era when all functional polymorphisms will be known.
Asunto(s)
Trastornos del Conocimiento/genética , ADN/genética , Inteligencia/genética , Proteínas del Tejido Nervioso/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo , Encéfalo/fisiología , Estudios de Casos y Controles , Niño , Preescolar , Pool de Genes , Pruebas Genéticas , Genoma Humano , Genotipo , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Valores de ReferenciaRESUMEN
The cell surface molecules controlling apoptosis in cortical neurons are largely unknown. A monoclonal antibody was derived that induces cultured neocortical neurons to undergo apoptosis. A Fab fragment of the antibody, however, lacked the ability to induce cell death. The antigen was purified, and characterized by compositional analysis, fast atom bombardment (FAB) mass spectrometry, sequential exoglycosidase treatments, methylation analysis, and (1)H-nuclear magnetic resonance spectroscopy, proving to be isoglobotetraosylceramide (IsoGb4). IsoGb4 has been shown previously to be a metastasis marker, antibodies against which block metastases in a mammary adenocarcinoma model (S. A. Carlsen et al., Cancer Res., 53: 2906-2911, 1993). Addition of the purified antigen to cells lacking this glycolipid demonstrated that it is capable of functioning as a portable apoptosis-transducing molecule. Intracellular ceramide levels were increased after the treatment with the apoptosis-inducing antibody, but the membrane sphingomyelin level remained unchanged. Fumonisin B1 inhibited both the ceramide increase and the apoptosis induced via IsoGb4, which indicated that the ceramide synthase pathway is likely to be involved in apoptosis induction by IsoGb4.
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Anticuerpos Monoclonales/metabolismo , Antígenos de Superficie/metabolismo , Apoptosis/fisiología , Globósidos/metabolismo , Neuronas/citología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Antígenos de Superficie/aislamiento & purificación , Apoptosis/inmunología , Secuencia de Carbohidratos , Transformación Celular Neoplásica , Globósidos/inmunología , Globósidos/aislamiento & purificación , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neuronas/inmunología , Neuronas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Both excitotoxicity and altered trophic factor support have been implicated in the pathogenesis of Alzheimer's disease. To determine whether stimulation of p75, the low-affinity receptor for nerve growth factor, contributes to the excitotoxin-induced apoptotic death of cholinergic neurons, we examined the effect of unilateral kainic acid (KA; PBS vehicle, 1.25, 2.5 and 5.0 nmol) administration into rat basal forebrain on neuronal loss and p75 expression. KA (2. 5 nmol) destroyed 43% of Nissl-stained neurons and 70% of choline acetyltransferase (ChAT)-positive neurons 5 days after injection. Agarose gel electrophoresis revealed that KA (2.5 nmol) induced local internucleosomal DNA fragmentation after 6-48 h. Immunohistochemical analysis further showed that KA (2.5 nmol) augmented p75 immunoreactivity at a time when terminal transferase-mediated deoxyuridine trophosphate (d-UTP)-digoxigenin nick end labeling (TUNEL)-positive nuclei were increased. Many fragmented nuclei were co-labeled with ChAT antibody. The chronic administration of anti-rat p75 or the protein synthesis inhibitor, cycloheximide, but not anti-human p75, substantially reduced the KA-induced destruction of cholinergic neurons and the induction of internucleosomal DNA fragmentation. Anti-rat p75, but not cycloheximide, also reversed the spatial memory impairment produced by KA. These findings suggest that overexpression of p75 contributes to the excitotoxin-induced death of rat basal forebrain cholinergic neurons by an apoptotic-like mechanism.
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Acetilcolina/metabolismo , Ácido Kaínico/farmacología , Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Prosencéfalo/efectos de los fármacos , Receptor de Factor de Crecimiento Nervioso/biosíntesis , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Recuento de Células/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Cicloheximida/farmacología , Fragmentación del ADN/efectos de los fármacos , Ácido Kaínico/administración & dosificación , Masculino , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/tratamiento farmacológico , Microinyecciones , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Prosencéfalo/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso/antagonistas & inhibidoresRESUMEN
The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.
Asunto(s)
Apoptosis/genética , Receptor de Factor de Crecimiento Nervioso/química , Receptor de Factor de Crecimiento Nervioso/metabolismo , Secuencia de Aminoácidos/genética , Animales , Sistema Libre de Células/metabolismo , Dimerización , Vectores Genéticos/genética , Humanos , Mutación/genética , Fragmentos de Péptidos/genética , Plásmidos/biosíntesis , Plásmidos/genética , Estructura Terciaria de Proteína/genética , Receptor de Factor de Crecimiento Nervioso/genética , Proteínas Recombinantes de Fusión/genética , Transfección , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismoRESUMEN
OBJECTIVES: Although most infants born prematurely do not have major developmental problems, those with perinatal medical problems and lengthy stays in the neonatal intensive care unit are at risk for sensory modulation problems and developmental sequelae. This study compared sensory responsiveness in preterm and full-term infants and examined the relationship of sensory responsiveness to temperament and developmental function. METHOD: Caregivers of infants with (n = 45) and without (n = 22) prematurity were asked to complete the Sensory Rating Scale. The preterm infants were also evaluated with the Bayley Scales of Infant Development, Second Edition (BSID-II). RESULTS: The preterm infants exhibited more frequent behaviors indicating tactile defensiveness and difficult temperament than did the full-term infants. When specific items were examined, the preterm infants displayed sensory-seeking behaviors and high activity levels. As measured by caregivers' report, sensory responsiveness was significantly related to temperament. It was not related to BSID-II Mental and Psychomotor scale scores. CONCLUSION: This study supports the findings of others that preterm infants have mild problems in sensory responsiveness and temperament. Correlational results do not support a definitive relationship between parents' reports of their infants' sensory responsiveness and developmental function.
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Recien Nacido Prematuro , Relaciones Padres-Hijo , Desempeño Psicomotor/fisiología , Temperamento/fisiología , Niño , Conducta Infantil , Desarrollo Infantil/fisiología , Femenino , Humanos , Recién Nacido , Masculino , Escalas de Valoración Psiquiátrica , Valores de Referencia , Reproducibilidad de los Resultados , Factores de Riesgo , Muestreo , Sensación , Sensibilidad y EspecificidadRESUMEN
Pyruvate is added to all media used for human in vitro fertilization and embryo culture, but its function(s) in the early embryo is unknown. We tested the possibility that pyruvate can act as an oxidizable energy source by measuring the consumption of pyruvate and oxygen by Day 2 and Day 3 human embryos, using microfluorometric techniques. Oxygen consumption (19.6 pmol/embryo per hour) could account for the oxidation of only 56% of the pyruvate consumed (13.9 pmol/embryo per hour). Oxygen was also consumed in the absence of exogenous substrates. Lactate appeared in the incubation medium with pyruvate (0.47 mM) as sole exogenous substrate at a rate of 12.1 pmol/embryo per hour, at a similar rate (10.85 pmol/embryo per hour) in the presence of 1 mM glucose and 0.47 mM pyruvate, and at 2.25 pmol/embryo per hour in the absence of exogenous substrates, suggesting that a high proportion of the pyruvate taken up by early human embryos is converted to lactate. Pyruvate uptake in the presence of UK5099, a pyruvate transport inhibitor, was reduced to 10% of control values, consistent with the presence of the monocarboxylate carrier in the human embryo plasma membrane.
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Embrión de Mamíferos/metabolismo , Ácido Pirúvico/metabolismo , Acrilatos/farmacología , Transporte Biológico/efectos de los fármacos , Medios de Cultivo , Técnicas de Cultivo , Glucosa/administración & dosificación , Humanos , Ácido Láctico/metabolismo , Oxidación-Reducción , Consumo de OxígenoRESUMEN
In order to develop another selective marker for cholinergic cell bodies and fibres, we have raised a highly specific polyclonal antibody against a peptide derived from the C-terminus of a recently cloned putative vesicular acetylcholine transporter. This antibody recognizes the vesicular acetylcholine transporter protein on western blots of membranes from transfected monkey fibroblast COS cells as well as from various rat brain regions but not from untransfected COS cells or rat liver. In separate mapping studies, the antibody was found to stain cell bodies and fibres in all of the regions of the nervous system known to be cholinergic, including (i) the various nuclei of the basal nuclear complex and their projections to the hippocampus, amygdala, and cerebral cortex, (ii) the caudate-putamen nucleus, accumbens nucleus, olfactory tubercle, and islands of Calleja complex, (iii) the medial habenula, (iv) the mesopontine cholinergic complex and its projections to the thalamus, extrapyramidal motor nuclei, basal forebrain, cingulate cortex, raphe and reticular nuclei, and some cranial nerve nuclei, and (v) the somatic motor and autonomic nuclei of the cranial and spinal nerves. In many of these cholinergic neurons, it is possible to detect immunoreactivity for the vesicular acetylcholine transporter in proximal portions of processes and their branches, as well as in numerous puncta in close association with them. Some of these puncta are large and surround cell bodies and processes of neurons in several regions, including the somatic motor neurons of cranial nerve nuclei in the brainstem and in the ventral horn of the spinal cord. Double immunofluorescence studies indicated that neurons positive for the vesicular acetylcholine transporter also stained for the biosynthetic enzyme of acetylcholine, choline acetyltransferase. We conclude that antibody against the C-terminus of the putative vesicular acetylcholine transporter provides another marker for cholinergic neurons that, unlike in situ hybridization procedures, labels terminals as well as cell bodies. Therefore this antibody has the potential to reveal changes in number and morphology of cholinergic cell bodies and their terminal varicosities that occur in both physiologic and pathologic conditions.
Asunto(s)
Acetilcolina/metabolismo , Proteínas Portadoras/metabolismo , Sistema Nervioso Central/metabolismo , Proteínas de Transporte de Membrana , Proteínas de Transporte Vesicular , Animales , Western Blotting , Química Encefálica , Línea Celular , Clonación Molecular , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Macaca mulatta , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
Emerging evidence suggests that the p75 neurotrophin receptor (p75NTR) mediates cell death; however, it is not known whether p75NTR negatively regulates other neuronal phenotypes. We found that mice null for p75NTR displayed highly significant increases in the size of basal forebrain cholinergic neurons, including those that are TrkA-positive. Cholinergic hippocampal target innervation also was increased significantly. Activity of the cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) was increased in both the medial septum and hippocampus. Upregulation of these cholinergic features was not associated with increased basal forebrain or hippocampal target NGF levels. In contrast, striatal cholinergic neurons, which do not express p75NTR, showed no difference in neuronal number, size, or ChAT activity between wild-type and p75NTR null mutant mice. These findings indicate that p75NTR negatively regulates cholinergic neuronal phenotype of the basal forebrain cholinergic neurons, including cell size, target innervation, and neurotransmitter synthesis.
Asunto(s)
Colina O-Acetiltransferasa/metabolismo , Hipocampo/patología , Neuronas/patología , Sistema Nervioso Parasimpático/patología , Prosencéfalo/metabolismo , Prosencéfalo/patología , Receptores de Factor de Crecimiento Nervioso/deficiencia , Animales , Recuento de Células , Cuerpo Estriado/patología , Dosificación de Gen , Hipocampo/metabolismo , Hipertrofia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Receptor de Factor de Crecimiento Nervioso , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Tabique Pelúcido/metabolismoRESUMEN
A role in neural development for protein tyrosine phosphatase (PTPase) receptors has been suggested by the finding of aberrant neurite outgrowth in Drosophila mutants lacking functional leukocyte common antigen-related (LAR) PTPase receptors; however, PTPase functions in the mammalian nervous system remain to be established. In transgenic mice containing a gene trap in the LAR gene, only trace expression of full-length LAR transcripts was found. In these mice, the size of basal forebrain cholinergic neurons was significantly reduced and cholinergic innervation of the dentate gyrus was markedly decreased. These findings constitute the first demonstration of an aberrant neuronal phenotype in a mammalian PTPase mutant and support the hypothesis that LAR-type PTPase receptors function to establish and/or maintain neuronal networks.
Asunto(s)
Giro Dentado/citología , Neuronas/fisiología , Proteínas Tirosina Fosfatasas/genética , Acetilcolina/fisiología , Animales , Benzoxazinas , Biomarcadores , Northern Blotting , Tamaño de la Célula/fisiología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/química , Fibras Colinérgicas/enzimología , Colorantes , Giro Dentado/química , Femenino , Expresión Génica/fisiología , Genotipo , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Neuronas/química , Neuronas/citología , Oxazinas , Fenotipo , Prosencéfalo/citología , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/análisis , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Receptores de Superficie Celular/genética , Receptores de Factor de Crecimiento Nervioso/análisisRESUMEN
The organization and distribution of the mRNA for the putative vesicular transporter for acetylcholine (VAChT) was studied in the rat brain by use of digoxigenin-labeled riboprobes and in situ hybridization technology. Signal was observed in all neural regions deduced to contain cholinergic somata on the basis of previous histochemical investigations employing choline acetyltransferase riboprobes and prior immunocytochemical studies with antibodies against choline acetyltransferase. It was absent in areas believed to contain no cholinergic neurons. Anti-sense riboprobes hybridized to the mRNA for the putative VAChT: (a) the projection neurons of the various nuclei of the basal nuclear complex, (b) the local circuit cells of the dorsal and ventral striata, (c) the projection neurons of the mesopontine complex, (d) perikarya in the ventral 2/3 of the medial habenula, (e) the somatic motor and autonomic cells of cranial nerves 3-7 and 9-12, as well as perikarya in the dorsal and ventral cochlear nuclei presumably giving rise to efferent fibers of cranial nerve 8, and (f) the alpha-motor and gamma-efferent motor neurons of the spinal cord. In addition, the mRNA for the VAChT was found in a few somata, probably ectopically located cells of the basal nuclear complex, in the internal capsule, central nucleus of the amygdala, entopeduncular nucleus, and zona incerta. It was also detected in some cell bodies in the reticular part of the substantia nigra, probably the rostral extension of the mesopontine complex, in the parabigeminal nucleus, and around the central canal in the spinal cord but not in cortical, hippocampal, and cerebellar perikarya. It is concluded that, like choline acetyltransferase, the mRNA for the putative acetylcholine vesicular transporter is another specific marker for neurons utilizing acetylcholine as a neurotransmitter. Further investigations of that transporter could have important implications for various diseases involving cholinergic systems, such as Alzheimer's disease.