RESUMEN
Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.
Asunto(s)
Edición Génica , Inmunodeficiencia Combinada Grave/genética , Animales , Proteína Quinasa Activada por ADN/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID , Proteínas Nucleares/genéticaRESUMEN
Mice lacking the intracellular glucocorticoid-regenerating enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) are protected from age-related spatial memory deficits. 11ß-HSD1 is expressed predominantly in the brain, liver and adipose tissue. Reduced glucocorticoid levels in the brain in the absence of 11ß-HSD1 may underlie the improved memory in aged 11ß-HSD1 deficient mice. However, the improved glucose tolerance, insulin sensitisation and cardioprotective lipid profile associated with reduced peripheral glucocorticoid regeneration may potentially contribute to the cognitive phenotype of aged 11ß-HSD1 deficient mice. In the present study, transgenic mice with forebrain-specific overexpression of 11ß-HSD1 (Tg) were intercrossed with global 11ß-HSD1 knockout mice (HSD1KO) to examine the influence of forebrain and peripheral 11ß-HSD1 activity on spatial memory in aged mice. Transgene-mediated delivery of 11ß-HSD1 to the hippocampus and cortex of aged HSD1KO mice reversed the improved spatial memory retention in the Y-maze but not spatial learning in the watermaze. Brain-derived neurotrophic factor (BDNF) mRNA levels in the hippocampus of aged HSD1KO mice were increased compared to aged wild-type mice. Rescue of forebrain 11ß-HSD1 reduced BDNF mRNA in aged HSD1KO mice to levels comparable to aged wild-type mice. These findings indicate that 11ß-HSD1 regenerated glucocorticoids in the forebrain and decreased levels of BDNF mRNA in the hippocampus play a role in spatial memory deficits in aged wild-type mice, although 11ß-HSD1 activity in peripheral tissues may also contribute to spatial learning impairments in aged mice.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/fisiología , Envejecimiento/psicología , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Terapia Genética , Trastornos de la Memoria/fisiopatología , Trastornos de la Memoria/terapia , Prosencéfalo/fisiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/deficiencia , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Envejecimiento/genética , Animales , Corticosterona/sangre , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Glucocorticoids (GCs) in utero influence embryonic development with consequent programmed effects on adult physiology and pathophysiology and altered susceptibility to cardiovascular disease. However, in viviparous species, studies of these processes are compromised by secondary maternal influences. The zebrafish, being fertilised externally, avoids this problem and has been used here to investigate the effects of transient alterations in GC activity during early development. Embryonic fish were treated either with dexamethasone (a synthetic GC), an antisense GC receptor (GR) morpholino (GR Mo), or hypoxia for the first 120h post fertilisation (hpf); responses were measured during embryonic treatment or later, post treatment, in adults. All treatments reduced cortisol levels in embryonic fish to similar levels. However, morpholino- and hypoxia-treated embryos showed delayed physical development (slower hatching and straightening of head-trunk angle, shorter body length), less locomotor activity, reduced tactile responses and anxiogenic activity. In contrast, dexamethasone-treated embryos showed advanced development and thigmotaxis but no change in locomotor activity or tactile responses. Gene expression changes were consistent with increased (dexamethasone) and decreased (hypoxia, GR Mo) GC activity. In adults, stressed cortisol values were increased with dexamethasone and decreased by GR Mo and hypoxia pre-treatments. Other responses were similarly differentially affected. In three separate tests of behaviour, dexamethasone-programmed fish appeared 'bolder' than matched controls, whereas Mo and hypoxia pre-treated fish were unaffected or more reserved. Similarly, the dexamethasone group but not the Mo or hypoxia groups were heavier, longer and had a greater girth than controls. Hyperglycaemia and expression of GC responsive gene (pepck) were also increased in the dexamethasone group. We conclude that GC activity controls many aspects of early-life growth and development in the zebrafish and that, like other species, manipulating GC status pharmacologically, physiologically or genetically in early life leads to programmable metabolic and behavioural traits in adulthood.
Asunto(s)
Conducta Animal/fisiología , Dexametasona/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Hiperglucemia/metabolismo , Pez Cebra/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Hidrocortisona/sangre , Hiperglucemia/genética , Hipoxia/genética , Hipoxia/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Pez Cebra/genéticaRESUMEN
Brain-derived neurotrophic factor (BDNF) signaling is implicated in the etiology of many psychiatric disorders associated with altered emotional processing. Altered peripheral (plasma) BDNF levels have been proposed as a biomarker for neuropsychiatric disease risk in humans. However, the relationship between peripheral and central BDNF levels and emotional brain activation is unknown. We used heterozygous BDNF knockdown rats (BDNF(+/-)) to examine the effects of genetic variation in the BDNF gene on peripheral and central BDNF levels and emotional brain activation as assessed by awake functional magnetic resonance imaging (fMRI). BDNF(+/-) and control rats were trained to associate a flashing light (conditioned stimulus; CS) with foot-shock, and brain activation in response to the CS was measured 24 h later in awake rats using fMRI. Central and peripheral BDNF levels were decreased in BDNF(+/-) rats compared with control rats. Activation of fear circuitry (amygdala, periaqueductal gray, granular insular) was seen in control animals; however, activation of this circuitry was absent in BDNF(+/-) animals. Behavioral experiments confirmed impaired conditioned fear responses in BDNF(+/-) rats, despite intact innate fear responses. These data confirm a positive correlation [r = 0.86, 95% confidence interval (0.55, 0.96); P = 0.0004] between peripheral and central BDNF levels and indicate a functional relationship between BDNF levels and emotional brain activation as assessed by fMRI. The results demonstrate the use of rodent fMRI as a sensitive tool for measuring brain function in preclinical translational studies using genetically modified rats and support the use of peripheral BDNF as a biomarker of central affective processing.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Condicionamiento Psicológico/fisiología , Miedo/fisiología , Aprendizaje/fisiología , Imagen por Resonancia Magnética , Amígdala del Cerebelo/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Condicionamiento Clásico/fisiología , Femenino , Imagen por Resonancia Magnética/métodos , Masculino , Estimulación Luminosa/métodos , Ratas Transgénicas , VigiliaRESUMEN
Glucocorticoid levels rise dramatically in late gestation to mature foetal organs in readiness for postnatal life. Immature heart function may compromise survival. Cardiomyocyte glucocorticoid receptor (GR) is required for the structural and functional maturation of the foetal heart in vivo, yet the molecular mechanisms are largely unknown. Here we asked if GR activation in foetal cardiomyocytes in vitro elicits similar maturational changes. We show that physiologically relevant glucocorticoid levels improve contractility of primary-mouse-foetal cardiomyocytes, promote Z-disc assembly and the appearance of mature myofibrils, and increase mitochondrial activity. Genes induced in vitro mimic those induced in vivo and include PGC-1α, a critical regulator of cardiac mitochondrial capacity. SiRNA-mediated abrogation of the glucocorticoid induction of PGC-1α in vitro abolished the effect of glucocorticoid on myofibril structure and mitochondrial oxygen consumption. Using RNA sequencing we identified a number of transcriptional regulators, including PGC-1α, induced as primary targets of GR in foetal cardiomyocytes. These data demonstrate that PGC-1α is a key mediator of glucocorticoid-induced maturation of foetal cardiomyocyte structure and identify other candidate transcriptional regulators that may play critical roles in the transition of the foetal to neonatal heart.
Asunto(s)
Corazón Fetal/fisiología , Glucocorticoides/farmacología , Mitocondrias/metabolismo , Miocitos Cardíacos/fisiología , Factores de Transcripción/fisiología , Animales , Regulación del Desarrollo de la Expresión Génica , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores de Glucocorticoides/metabolismo , Transducción de SeñalAsunto(s)
Ansiedad/metabolismo , Expresión Génica , Glucógeno Sintasa Quinasa 3/biosíntesis , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Neurregulina-1/biosíntesis , Estrés Psicológico/metabolismo , Envejecimiento/psicología , Animales , Femenino , Glucógeno Sintasa Quinasa 3 beta , Masculino , Ratones , Factores de TiempoRESUMEN
Foetal growth restriction (FGR), reflective of an adverse intrauterine environment, confers a significantly increased risk of perinatal mortality and morbidity. In addition, low birthweight associates with adult diseases including hypertension, metabolic dysfunction and behavioural disorders. A key mechanism underlying FGR is exposure of the foetus to glucocorticoids which, while critical for foetal development, in excess can reduce foetal growth and permanently alter organ structure and function, predisposing to disease in later life. Foetal glucocorticoid exposure is regulated, at least in part, by the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which catalyses the intracellular inactivation of glucocorticoids. This enzyme is highly expressed within the placenta at the maternal-foetal interface, limiting the passage of glucocorticoids to the foetus. Expression of 11ß-HSD2 is also high in foetal tissues, particularly within the developing central nervous system. Down-regulation or genetic deficiency of placental 11ß-HSD2 is associated with significant reductions in foetal growth and birth weight, and programmed outcomes in adulthood. To unravel the direct significance of 11ß-HSD2 for developmental programming, placental function, neurodevelopment and adult behaviour have been extensively investigated in a mouse knockout of 11ß-HSD2. This review highlights the evidence obtained from this mouse model for a critical role of feto-placental 11ß-HSD2 in determining the adverse programming outcomes.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Desarrollo Fetal/fisiología , Glucocorticoides/metabolismo , Placenta/metabolismo , Animales , Femenino , Retardo del Crecimiento Fetal/etiología , Retardo del Crecimiento Fetal/metabolismo , Feto , Glucocorticoides/efectos adversos , Síndrome Metabólico/embriología , Síndrome Metabólico/metabolismo , Ratones , EmbarazoRESUMEN
An imbalance between central glucocorticoid (GR) and mineralocorticoid (MR) receptors is proposed to underlie the HPA axis dysregulation that associates with susceptibility to psychopathology (anxiety, PTSD). To test this 'balance hypothesis' we examined whether the impact of MR levels upon HPA-axis control and behaviour depended on the relative levels of GR and vice versa. Avoiding antenatal maternal 'programming' effects by using littermates, we generated mice with forebrain MR over-expression (MR(hi)) and/or simultaneous global GR under-expression (GR(lo)). We found a significant interaction between MR and GR in control of the HPA-axis under stressed but not basal conditions. With reduced GR levels, HPA-axis activity in response to restraint stress was enhanced, likely due to impaired negative feedback. However, high MR in concert with reduced GR minimised this HPA-axis overshoot in response to stress. MR:GR balance also played a role in determining strategies of spatial memory during a watermaze probe trial: when coupled with GR under-expression, MR(hi) show enhanced perseveration, suggesting enhanced spatial recall or reduced exploratory flexibility. Other alterations in cognitive functions were specific to a single receptor without interaction, with both MR(hi) and GR(lo) manipulations independently impairing reversal learning in spatial and fear memory tasks. Thus, MR and GR interact in specific domains of neuroendocrine and cognitive control, but for other limbic-associated behaviours each receptor mediates its own repertoire of responses. Since modulation of HPA-axis and behavioural dysfunction associated with high levels of MR, selective ligands or transcriptional regulators may afford novel therapeutic approaches to affective psychopathologies.
Asunto(s)
Conducta Animal/fisiología , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Animales , Cognición/fisiología , Regulación de la Expresión Génica/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Sistema Hipotálamo-Hipofisario/fisiopatología , Masculino , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Sistema Hipófiso-Suprarrenal/fisiopatología , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Restricción Física/fisiologíaRESUMEN
There is increasing evidence for a role for epigenetic modifications in early life 'programming' effects. Altered placental methyl donor transport may impact on the establishment of epigenetic marks in the fetus. This study investigated the effects of prenatal glucocorticoid overexposure on placental methyl donor transport. Glucocorticoids increased folate but decreased choline transport and reduced fetal plasma methionine levels. There was no change in global DNA methylation in fetal liver. These data suggest prenatal glucocorticoid overexposure causes complex alterations in the placental transport of key methyl donors which may have important implications for maternal diet and nutrient supplementation in pregnancy.
Asunto(s)
Dexametasona/farmacología , Desarrollo Embrionario/efectos de los fármacos , Metano/análogos & derivados , Placenta/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Colina/farmacocinética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/fisiología , Dexametasona/efectos adversos , Desarrollo Embrionario/genética , Epistasis Genética/efectos de los fármacos , Epistasis Genética/genética , Femenino , Ácido Fólico/farmacocinética , Metano/metabolismo , Metionina/farmacocinética , Placenta/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/metabolismo , Ratas , Ratas WistarRESUMEN
A dissipative particle dynamics computer simulation is used to investigate the ability of small oil molecules (hexane, dodecane, and octadecane) to control phase structures in nonionic surfactant-water systems. The model is successfully tested against the experimental results for binary and ternary systems where the third components are "swelling" and "penetrating" oils. The experimentally observed phases present in such systems were successfully modeled. In addition, the simulations show the locations of the oil molecules within the bilayer and the surfactant chain conformation. While the simulations confirm much of what is expected from experiment and theoretical models, evidence is found for the terminal methyl end of the surfactant molecules being located slightly closer to the interfacial region than other groups in the same chain.
Asunto(s)
Tensoactivos/química , Agua/química , Alcanos/química , Simulación por Computador , Hexanos/química , Modelos TeóricosRESUMEN
Low birth weight in humans is predictive of hypertension in adult life, and while the mechanisms underlying this link remain unknown, fetal overexposure to glucocorticoids has been implicated. We have previously shown that prenatal dexamethasone (DEX) exposure in the rat lowers birth weight and programmes adult hypertension. This current study aimed to unravel the molecular nature of this hypertension. However, unknowingly, post hoc investigations revealed that our animals had been subjected to environmental noise stresses from an adjacent construction site, which were sufficient to confound our prenatal DEX-programming experiments. This perinatal stress successfully established low birth weight, hypercorticosteronaemia, insulin resistance, hypertension and hypothalamic-pituitary-adrenal axis dysfunction in vehicle (VEH)-treated offspring, such that the typical distinctions between both treatment groups were ameliorated. The lack of an additional effect on DEX-treated offspring is suggestive of a maximal effect of perinatal stress and glucocorticoids, serving to prevent against the potentially detrimental effects of sustained glucocorticoid hyper-exposure. Finally, this paper serves to inform researchers of the potential detrimental effects of neighbouring construction sites to their experiments.
Asunto(s)
Dexametasona/toxicidad , Ambiente , Glucocorticoides/toxicidad , Hipertensión/etiología , Ruido/efectos adversos , Efectos Tardíos de la Exposición Prenatal/etiología , Animales , Animales Recién Nacidos , Peso al Nacer/efectos de los fármacos , Peso al Nacer/fisiología , Corticosterona/sangre , Femenino , Prueba de Tolerancia a la Glucosa , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/fisiopatología , Resistencia a la Insulina , Masculino , Exposición Materna , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/fisiopatología , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Ratas , Ratas Wistar , Estrés FisiológicoRESUMEN
Inter-individual differences in hypothalamic-pituitary-adrenal (HPA) axis activity underlie differential vulnerability to neuropsychiatric and metabolic disorders, although the basis of this variation is poorly understood. 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has previously been shown to influence HPA axis activity. 129/MF1 mice null for 11beta-HSD1 (129/MF1 HSD1(-/-)) have greatly increased adrenal gland size and altered HPA activity, consistent with reduced glucocorticoid negative feedback. On this background, concentrations of plasma corticosterone and adrenocorticotrophic hormone (ACTH) were elevated in unstressed mice, and showed a delayed return to baseline after stress in HSD1-null mice with reduced sensitivity to exogenous glucocorticoid feedback compared to same-background genetic controls. In the present study, we report that the genetic background can dramatically alter this pattern. By contrast to HSD1(-/-) mice on a 129/MF1 background, HSD1(-/-) mice congenic on a C57Bl/6J background have normal basal plasma corticosterone and ACTH concentrations and exhibit normal return to baseline of plasma corticosterone and ACTH concentrations after stress. Furthermore, in contrast to 129/MF1 HSD1(-/-) mice, C57Bl/6J HSD1(-/-) mice have increased glucocorticoid receptor expression in areas of the brain involved in glucocorticoid negative feedback (hippocampus and paraventricular nucleus), suggesting this may be a compensatory response to normalise feedback control of the HPA axis. In support of this hypothesis, C57Bl/6J HSD1(-/-) mice show increased sensitivity to dexamethasone-mediated suppression of peak corticosterone. Thus, although 11beta-HSD1 appears to contribute to regulation of the HPA axis, the genetic background is crucial in governing the response to (and hence the consequences of) its loss. Similar variations in plasticity may underpin inter-individual differences in vulnerability to disorders associated with HPA axis dysregulation. They also indicate that 11beta-HSD1 inhibition does not inevitably activate the HPA axis.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Sistema Hipotálamo-Hipofisario , Sistema Hipófiso-Suprarrenal , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Glándulas Suprarrenales/patología , Hormona Adrenocorticotrópica/sangre , Animales , Secuencia de Bases , Ritmo Circadiano , Corticosterona/sangre , Cartilla de ADN , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Reacción en Cadena de la PolimerasaRESUMEN
Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR(betageo/+) mice were generated from embryonic stem (ES) cells with a gene trap integration of a beta-galactosidase-neomycin phosphotransferase (betageo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GR(betageo/+) mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin-aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GR(betageo/+) mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GR(betageo/+) mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Dieta/efectos adversos , Grasas de la Dieta/efectos adversos , Hipertensión/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Glucocorticoides/metabolismo , Adiposidad/efectos de los fármacos , Adiposidad/genética , Aldosterona/metabolismo , Angiotensinas/metabolismo , Animales , Glucemia/metabolismo , Línea Celular , Grasas de la Dieta/farmacología , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Humanos , Hipertensión/inducido químicamente , Hipertensión/genética , Metabolismo de los Lípidos/genética , Ratones , Ratones Transgénicos , Receptores de Glucocorticoides/genética , Renina/metabolismo , Aumento de Peso/efectos de los fármacos , Aumento de Peso/genéticaRESUMEN
BACKGROUND: Circadian rhythms control a vast array of biological processes in a broad spectrum of organisms. The contribution of circadian rhythms to the development of megakaryocytes and the regulation of platelet biology has not been defined. OBJECTIVES: This study tested the hypothesis that murine megakaryocytes exhibit hallmarks of circadian control. METHODS: Mice expressing a PER2::LUCIFERASE circadian reporter protein and C57BI/6 mice were used to establish if megakaryocytes expressed circadian genes in vitro and in vivo. Mice were also subjected to 3 weeks on a restricted feeding regime to separate food-entrained from light-entrained circadian rhythms. Quantitative real time polymerase chain reaction (PCR), flow cytometry and imunohistochemistry were employed to analyse gene expression, DNA content and cell-cycle behavior in megakaryocytes collected from mice over a 24-h period. RESULTS: Megakaryocytes exhibited rhythmic expression of the clock genes mPer2 and mBmal1 and circadian rhythms in megakaryopoiesis. mPer2 and mBmal1 expression phase advanced 8 h to coincide with the availability of food; however, food availability had a more complex effect on megakaryopoiesis, leading to a significant overall increase in megakaryocyte ploidy levels and cell-cycle activity. CONCLUSIONS: Normal megakaryopoiesis requires synchrony between food- and light-entrained circadian oscillators.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Ingestión de Alimentos/fisiología , Eritropoyesis , Megacariocitos/citología , Proteínas Nucleares/genética , Periodicidad , Factores de Transcripción/genética , Factores de Transcripción ARNTL , Animales , Ciclo Celular , Ritmo Circadiano/genética , Ingestión de Alimentos/genética , Alimentos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Proteínas Circadianas PeriodRESUMEN
Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.
Asunto(s)
Desoxirribonucleasas/genética , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , Ingeniería Genética/métodos , Genoma Humano/genética , Dedos de Zinc/genética , Secuencia de Bases , Estudios de Evaluación como Asunto , Humanos , Datos de Secuencia MolecularRESUMEN
11beta-Hydroxysteroid dehydrogenase type 2 is a glucocorticoid metabolizing enzyme that catalyzes rapid inactivation of corticosterone and cortisol to inert 11-keto derivatives. As 11beta-hydroxysteroid dehydrogenase type 2 is highly expressed in the developing brain, but not in the adult CNS, we hypothesized that it may represent a protective barrier to the deleterious actions of corticosteroids on proliferating cells. To test this hypothesis we have investigated the development and growth of the cerebellum in neonatal C57BL/6 mice and mice lacking 11beta-hydroxysteroid dehydrogenase type 2 (-/-). 11beta-Hydroxysteroid dehydrogenase type 2-/- mice had consistently lower body weight throughout the neonatal period, coupled with a smaller brain size although this was normalized when corrected for body weight. The cerebellar size was smaller in 11beta-hydroxysteroid dehydrogenase type 2-/- mice, due to decreases in size of both the molecular and internal granule layers. When exogenous corticosterone was administered to the pups between postnatal days 4 and 13, 11beta-hydroxysteroid dehydrogenase type 2(-/-) mice were more sensitive, showing further inhibition of cerebellar growth while the wildtype mice were not affected. Upon withdrawal of exogenous steroid, there was a rebound growth spurt so that at day 21 postnatally, the cerebellar size in 11beta-hydroxysteroid dehydrogenase type 2-/- mice was similar to untreated mice of the same genotype. Furthermore, 11beta-hydroxysteroid dehydrogenase type 2-/- mice had a delay in the attainment of neurodevelopmental landmarks such as negative geotaxis and eye opening. We therefore suggest that 11beta-hydroxysteroid dehydrogenase type 2 acts as to protect the developing nervous system from the deleterious consequences of glucocorticoid overexposure.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/fisiología , Animales Recién Nacidos/fisiología , Cerebelo/fisiología , Glucocorticoides/fisiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Animales , Peso Corporal/fisiología , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Proliferación Celular , Cerebelo/crecimiento & desarrollo , Cerebelo/patología , Corticosterona/sangre , Femenino , Proteína Ácida Fibrilar de la Glía/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/fisiología , Equilibrio Postural/fisiología , Reflejo/fisiologíaRESUMEN
Glucocorticoid overexposure in utero may underlie the association between low birth weight and subsequent development of common cardiovascular and metabolic pathologies. Previously, we have shown that prenatal dexamethasone (DEX) exposure in rat reduces birth weight and programs the hypothalamic-pituitary axis and fasting and postprandial hyperglycemia in adult males and hypertension in adult males and females. This study aimed to determine 1) whether there were gender differences in prenatal DEX-programmed offspring, and 2) whether the renin-angiotensin system (RAS) plays a role in the programming of hypertension. Rats exposed to DEX in utero (100 microg.kg(-1).day(-1) from embryonic days 14-21) were of lower birth weight (by 12%, P < 0.01) and displayed full catch-up growth within the first month of postnatal life. DEX-treated male offspring in adulthood selectively displayed elevated plasma adrenocorticotropic hormone (by 221%) and corticosterone (by 188%, P < 0.05), postprandial insulin-glucose ratios (by 100%, P < 0.05), and hepatic expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (by 38%, P < 0.05). Conversely, DEX-programmed females were hypertensive (by 11%, P < 0.05), with elevated hepatic angiotensinogen mRNA expression (by 9%, P < 0.05), plasma angiotensinogen (by 61%, P < 0.05), and renin activity (by 88%, P < 0.05). These findings demonstrate that prenatal glucocorticoids program adulthood cardiovascular and metabolic physiology in a gender-specific pattern, and that an activated RAS may in part underlie the hypertension associated with prenatal DEX programming.
Asunto(s)
Glucocorticoides/farmacología , Intolerancia a la Glucosa/fisiopatología , Hipertensión/fisiopatología , Efectos Tardíos de la Exposición Prenatal , Sistema Renina-Angiotensina/efectos de los fármacos , Hormona Adrenocorticotrópica/sangre , Hormona Adrenocorticotrópica/efectos de los fármacos , Animales , Peso al Nacer/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Corticosterona/sangre , Dexametasona/farmacología , Femenino , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/fisiopatología , Masculino , Fenotipo , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/fisiopatología , Embarazo , Ratas , Ratas Wistar , Caracteres SexualesRESUMEN
Magnocellular neurosecretory neurones in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei express oestrogen receptor beta (ERbeta) but not ERalpha. In the PVN, ERbeta is strongly expressed in the ventromedial parvocellular neurones projecting to the brainstem. We used quantitative in situ hybridization, with (35)S-labelled riboprobes, to study heterologous regulation by manipulating adrenal steroid hormones (72 h after adrenalectomy +/- corticosterone replacement; repeated stress: halothane inhalation, environmental cold, immobilization, each daily for 3 days) in male rats. Adrenalectomy increased ERbeta mRNA expression in the magnocellular PVN and SON, by 2.2 and 2.5-fold, respectively, with no effect in the ventromedial parvocellular PVN neurones. Corticosterone replacement partially prevented the increases in ERbeta mRNA expression in magnocellular PVN and SON neurones. Repeated stress over 72 h had no effect on ERbeta mRNA expression in the magnocellular PVN or SON, but increased expression 1.4-fold in the ventromedial parvocellular PVN neurones. Although consequences of hydromineral balance derangement after adrenalectomy may stimulate magnocellular neurones, strongly stimulating the neurones by giving intact male rats 2% saline to drink for 72 h decreased ERbeta mRNA expression in the magnocellular PVN and SON neurones by approximately 60%, and in the ventromedial parvocellular PVN neurones by 13%. Thus, ERbeta mRNA expression is negatively regulated by basal glucocorticoid secretion in magnocellular PVN and SON neurones, and positively regulated by stress in ventromedial parvocellular PVN neurones. However, ERbeta mRNA expression in magnocellular neurones is negatively linked to hyperosmotic stimulation of the neurones. The 6.25-fold variation in ERbeta mRNA expression in magnocellular neurones from salt-loading to adrenalectomy could alter their sensitivity to oestrogens. Consequently, regulation of oxytocin and vasopressin neurone activity via ERbeta is expected to vary according to their functional state and, in particular, on basal glucocorticoid actions.
Asunto(s)
Corticosterona/fisiología , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores de Estrógenos/genética , Estrés Fisiológico/metabolismo , Núcleo Supraóptico/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Adaptación Fisiológica , Adrenalectomía , Análisis de Varianza , Animales , Receptor beta de Estrógeno , Regulación de la Expresión Génica , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Sistema Hipófiso-Suprarrenal/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Receptores de Estrógenos/metabolismo , Solución Salina Hipertónica , Estrés Fisiológico/fisiopatología , Núcleo Supraóptico/citologíaRESUMEN
Nitric oxide synthase (NOS) has recently been shown to be an important pathophysiological regulator in experimental implantation glioma since manipulation of NOS can significantly alter tumoural blood flow and inhibit tumour growth. In this study we investigated the role of iNOS (inducible NOS) in glioma tumourogenisis using the rodent C6 striatal implantation model. We produced genetically engineered C6 clones that do not express iNOS activity even after stimulation with a mixture of lipopolysaccaride (LPS) and tumour necrosis factor (TNF)-alpha. These iNOS knockout cells showed a similar growth rate to control cells in vivo at 5 days. We then performed an in vivo implantation glioma study using either the iNOS knockout clone or two genetically engineered control C6 clones. There was a significant reduction (p < 0.01) of tumour mass with the iNOS knockout clone 28 days after the implantation. Immunocytochemistry indicated infiltrates of CD3 positive T cells and macrophages in the controls and the iNOS knockout group. These studies indicate that iNOS expression by tumour parenchymal cells is a critical factor for tumour growth with this model. The mechanisms that cause failure of tumour growth need clarification prior to considering that specific iNOS inhibitors might be candidates for adjuvant treatment of malignant glioma.