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BACKGROUND: Resolution of inflammation is an active process involving specialised pro-resolving mediators (SPMs) generated from the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-3 fatty acid supplementation during infancy may provide an intervention strategy to modify SPMs and reduce oxidative stress. This study evaluates the effect of omega-3 fatty acid supplementation in infancy on SPMs and F2-isoprostanes from 6 months to 5 years of age. METHODS: In a double-blind, placebo-controlled, parallel-group study design, 420 infants were randomized to a daily supplement of omega-3 fatty acids (280mg DHA and 110mg EPA) or olive oil (control), from birth to age 6 months. Blood was collected at birth (cord blood), 6 months, 12 months and 5 years. Plasma SPMs included 18-HEPE, E-series resolvins, 17-HDHA, D-series resolvins, 14-HDHA, 10S,17S-DiHDoHE, MaR1 and PD1. F2-isoprostanes were measured in plasma and urine, as markers of oxidative stress in vivo. RESULTS: The change in the concentration of 18-HEPE from birth to 6 months was greater in the omega-3 fatty acid group (Ptimepoint*group=0.04) with levels at 6 months significantly higher than controls (P=0.02). Other SPMs were not different between the groups at any time point. Plasma 18-HEPE concentration were associated with erythrocyte EPA concentrations after age and group adjustments (P<0.001), but not with allergic outcomes at 12 months. There were no between-group differences in plasma and urinary F2-isoprostanes at any time point. CONCLUSION: Omega-3 fatty acid supplementation from birth to 6 months of age increased SPM at 6 months but the effects were not sustained after supplementation ceased. Given that 18-HEPE is a biologically active metabolite, future studies should examine how the increase in 18-HEPE relates to potential health benefits of omega-3 fatty acid supplementation in infancy.
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Biomarcadores/sangre , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Hidroxieicosatetraenoicos/sangre , Inflamación/sangre , Preescolar , Suplementos Dietéticos/efectos adversos , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/sangre , Ácido Eicosapentaenoico/administración & dosificación , Ácido Eicosapentaenoico/sangre , Ácidos Grasos Omega-3/efectos adversos , Ácidos Grasos Omega-3/sangre , Femenino , Humanos , Lactante , Inflamación/fisiopatología , Masculino , Aceite de Oliva/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , EmbarazoRESUMEN
Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1ß and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity.
RESUMEN
BACKGROUND: Oxidative stress and nutritional deficiency may influence the excessive shortening of the telomeric ends of chromosomes. It is known that stress exposure in intrauterine life can produce variations in telomere length (TL), thereby potentially setting up a long-term trajectory for disease susceptibility. OBJECTIVE: To assess the effect of omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) supplementation during pregnancy on telomere length and oxidative stress in offspring at birth and 12 years of age (12y). DESIGN: In a double-blind, placebo-controlled, parallel-group study, 98 pregnant atopic women were randomised to 4g/day of n-3 LCPUFA or control (olive oil [OO]), from 20 weeks gestation until delivery. Telomere length as a marker of cell senescence and plasma and urinary F2-isoprostanes as a marker of oxidative stress were measured in the offspring at birth and 12y. RESULTS: Maternal n-3 LCPUFA supplementation did not influence offspring telomere length at birth or at 12y with no changes over time. Telomere length was not associated with F2-isoprostanes or erythrocyte total n-3 fatty acids. Supplementation significantly reduced cord plasma F2-isoprostanes (P<0.001), with a difference in the change over time between groups (P=0.05). However, the differences were no longer apparent at 12y. Between-group differences for urinary F2-isoprostanes at birth and at 12y were non-significant with no changes over time. CONCLUSIONS: This study does not support the hypothesis that n-3 LCPUFA during pregnancy provides sustained effects on postnatal oxidative stress and telomere length as observed in the offspring.
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F2-Isoprostanos/sangre , F2-Isoprostanos/orina , Ácidos Grasos Omega-3/administración & dosificación , Telómero/efectos de los fármacos , Niño , Suplementos Dietéticos , Método Doble Ciego , Eritrocitos/química , Ácidos Grasos Omega-3/farmacología , Femenino , Humanos , Estrés Oxidativo/efectos de los fármacos , Embarazo , Atención PrenatalRESUMEN
Therapies targeting cancer metastasis are challenging owing to the complexity of the metastatic process and the high number of effectors involved. Although tumour hypoxia has previously been associated with increased aggressiveness as well as resistance to radio- and chemotherapy, the understanding of a direct link between the level and duration of hypoxia and the individual steps involved in metastasis is still missing. Using live imaging in a chick embryo model, we have demonstrated that the exposure of neuroblastoma cells to 1% oxygen for 3 days was capable of (1) enabling cell migration towards blood vessels, (2) slowing down their velocity within blood vessels to facilitate extravasation and (3) promoting cell proliferation in primary and secondary sites. We have shown that cells do not have to be hypoxic anymore to exhibit these acquired capabilities as a long-term memory of prior hypoxic exposure is kept. Furthermore, non-hypoxic cells can be influenced by neighbouring hypoxic preconditioned cells and be entrained in the metastatic progression. The acquired aggressive phenotype relies on hypoxia-inducible factor (HIF)-dependent transcription of a number of genes involved in metastasis and can be impaired by HIF inhibition. Altogether, our results demonstrate the need to consider both temporal and spatial tumour heterogeneity because cells can 'remember' an earlier environment and share their acquired phenotype with their close neighbours. As a consequence, it is necessary to monitor the correct hypoxic markers to be able to predict the consequences of the cells' history on their behaviour and their potential response to therapies.
RESUMEN
Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gland. Tumours that occur in patients under 18 months of age have a particularly good prognosis and frequently undergo spontaneous regression. This led to the hypothesis that developmental cues in the youngest patients may prompt belated differentiation and/or apoptosis of the tumour cells. To test our hypothesis, we have injected MYCN-amplified neuroblastoma cells into the extra embryonic veins of chick embryos at embryonic day 3 (E3) and E6 and analysed the response of these Kelly cells at E10 and E14. Amplification of the MYCN gene occurs in up to 30% of tumours and is normally associated with a very poor prognosis. Kelly cells injected at E3 follow neural crest pathways and integrate into neural locations such as SG and the enteric nervous system although never into the adrenal gland. Additionally they migrate to non-neural locations such as the heart, meninges, jaw regions and tail. The cells respond to their respective microenvironments and in SG, some cells differentiate, they show reduced cell division and crucially all cells have undetectable MYCN expression by E10. In non-neural locations, cells form more rapidly dividing clumps and continue to express MYCN. The downregulation of MYCN is dependent on continuous and direct interaction with the sympathetic ganglion environment. We propose that the MYCN-amplicon in the Kelly cells retains the ability to correctly interpret the environmental cues leading to downregulation of MYCN.
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Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB cell lines, we have demonstrated that the chemotherapeutic drug etoposide induces a p53- and caspase-dependent cell death. We have observed an additional caspase-independent cell death mechanism involving delayed nuclear factor κB (NF-κB) activity. The delayed induction was controlled by a p53-dependent transcription step and the production of death receptors (especially CD95/Fas). We further demonstrated that in both MB and glioblastoma (GM) cell lines, in which the p53 pathway was not functional, no p65 activation could be detected upon etoposide treatment. MB cell lines that have mutations in p53 or NF-κB are either less sensitive (NF-κB mutant) or even completely resistant (p53 mutant) to chemotherapeutic intervention. The optimal cell death was only achieved when both p53 and NF-κB were switched on. Taken together, our results shed light on the mechanism of NF-κB activation by etoposide in brain tumours and show that the genetic background of MB and GM cells determines their sensitivity to chemotherapy and has to be taken into account for efficient therapeutic intervention.
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Neoplasias Cerebelosas/patología , Etopósido/farmacología , Meduloblastoma/patología , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Cerebelosas/enzimología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Meduloblastoma/enzimología , Modelos Biológicos , Fosforilación/efectos de los fármacos , Receptores de Muerte Celular/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
Oscillations in second-messenger signalling (e.g. calcium) have previously been shown to be important in the control of transcription. More recently, oscillations in localization and absolute levels of transcription factors and their regulators have been identified. Here we discuss the role of network motifs such as the negative feedback loop and their role in oscillatory signalling, and how oscillations in components of the nuclear factor kappaB signalling pathway are important to the dynamic control of transcription in response to a cytokine stimulus.
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Regulación de la Expresión Génica/fisiología , Factores de Transcripción/metabolismo , Animales , Retroalimentación , Cinética , Modelos Genéticos , FN-kappa B/fisiología , OscilometríaRESUMEN
Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.
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Regulación de la Expresión Génica , FN-kappa B/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Simulación por Computador , Citoplasma/metabolismo , Etopósido/farmacología , Retroalimentación Fisiológica , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Modelos Biológicos , Inhibidor NF-kappaB alfa , Fosforilación , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción ReIA , Transcripción Genética , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Reactive oxygen species (ROS) cause death of cerebellar granule neurons. Here, a 15-min pulse of H(2)O(2) (100 microm) induced an active process of neuronal death distinct from apoptosis. Oxidative stress activated a caspase-independent but calpain-dependent decline of calcium/calmodulin-dependent protein kinase IV and cAMP- responsive element-binding protein (CREB). Calpain inhibitors restored calcium/calmodulin-dependent protein kinase IV and CREB but did not influence phosphorylated CREB levels or survival, indicating recruitment of an additional dephosphorylation process. Co-treatment with calpain and serine/threonine phosphatase inhibitors restored pCREB levels and rescued neurons. This phosphatase-activated signaling pathway was shown to be dependent on de novo protein synthesis. Further, gene transfer studies revealed that CREB is a common final effector of both apoptosis and ROS-induced death. Our data indicate that dephosphorylation and proteolytic signaling mechanisms underlie ROS-induced programmed cell death.
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Apoptosis , Calpaína/fisiología , Neuronas/patología , Estrés Oxidativo , Fosfoproteínas Fosfatasas/fisiología , Animales , Calcineurina/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Caspasas/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/análisis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Peróxido de Hidrógeno/toxicidad , Ratones , Necrosis , Fosforilación , Biosíntesis de Proteínas , Serina/metabolismoRESUMEN
The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor, a seven-domain transmembrane receptor, is positively coupled to both adenylate cyclase and phospholipase C. PACAP exerts neurotrophic effects which are mainly mediated through the cAMP/protein kinase A pathway. Here we show that the cell-permeable C2-ceramide selectively blocks PACAP-activated cAMP production, without affecting phosphoinositide breakdown. Thus by blocking the neuroprotective cAMP signalling pathway, C2-ceramide will reinforce its direct death-inducing signalling. We found that a reactive oxygen species scavenger reversed the C2-ceramide effect and that H2O2 mimicked it. Together these data indicate that reactive oxygen species (ROS) mediates C2-ceramide-induced cAMP pathway uncoupling. This uncoupling did not involve ATP supply or Galphas protein function but rather adenylate cyclase function per se. Further, the tyrosine phosphatase inhibitors, but not the serine/threonine phosphatase inhibitors, prevent inhibition of cAMP production by ROS. This suggests that H2O2 requires a functional tyrosine phosphatase(s) to block PACAP-dependent cAMP production.
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AMP Cíclico/fisiología , Neuropéptidos/farmacología , Especies Reactivas de Oxígeno/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Esfingosina/análogos & derivados , Esfingosina/fisiología , Adenilil Ciclasas/fisiología , Línea Celular , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/biosíntesis , Proteínas de Unión al GTP/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Proteínas Tirosina Fosfatasas/fisiología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/fisiología , Esfingosina/farmacologíaRESUMEN
The neuroprotective mechanisms of the Ca2+/calmodulin kinase (CaMK) signaling pathway were studied in primary cerebellar neurons in vitro. When switched from depolarizing culture conditions HK (extracellular K+ 30 mM) to LK (K+ 5 mM), these neurons rapidly undergo nuclear fragmentation, a typical feature of apoptosis. We present evidence that blockade of L-type Ca2+ channels (nifedipine sensitive) but not N/P/Q-type Ca2+ channels (omega-conotoxin MVIIC sensitive) triggered apoptosis and CPP32/caspase-3-like activity. The entry into apoptosis was associated with a progressive caspase-3-dependent cleavage of CaMKIV, but not of CaMKII. CaMKIV function in neuronal apoptosis was further investigated by overexpression of CaMKIV mutants by gene transfer. A dominant-active CaMKIV mutant inhibited LK-induced apoptosis whereas a dominant-negative form induced apoptosis in HK, suggesting that CaMKIV exerts neuroprotective effects. The transcription factor CREB is a well-described nuclear target of CaMKIV in neurons. When switched to LK, the level of phosphorylation of CREB, after an initial drop, further declined progressively with kinetics comparable to those of CaMKIV degradation. This decrease was abolished by caspase-3 inhibitor. These data are compatible with a model where Ca2+ influx via L-type Ca2+ channels prevents caspase-dependent cleavage of CaMKIV and promotes neuronal survival by maintaining a constitutive level of CaMKIV/CREB-dependent gene expression.
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Apoptosis/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cerebelo/efectos de los fármacos , Neuronas/efectos de los fármacos , Potasio/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Señalización del Calcio/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/enzimología , Cerebelo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dihidropiridinas/agonistas , Dihidropiridinas/antagonistas & inhibidores , Dihidropiridinas/farmacología , Genes Dominantes , Ratones , Modelos Biológicos , Mutación , Neuronas/citología , Neuronas/enzimología , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Nifedipino/farmacología , Fosforilación , Potasio/administración & dosificación , Procesamiento Proteico-PostraduccionalRESUMEN
OBJECTIVE: Prescription of opiates to non cancer chronic pain patients is controversial, partly because of the risk of tolerance and dependence development. The two objectives of that review were: a) to identify the factors which may explain the variability of tolerance and dependence in clinical practice; b) to analyse the cellular mechanisms of occurrence of those phenomenons. DATA SOURCES AND EXTRACTION: To our own file, we added articles retrieved in the Medline database, using, alone or in combination, following key-words (opiate, tolerance, dependence, opiate receptor, pain treatment, cAMP, cGMP, NO, NMDA, protein kinase, gene). Out of nearly 450 articles, we selected less than 200. DATA SYNTHESIS: Tolerance, defined as loss of opioid efficacy with time, is extremely variable and depends on pain mechanisms, intrinsic efficacy and administration modality of the opioid, as well as co-administration of other agents. Physical dependence is a consequence of the intrinsic and extrinsic adaptations concerning structures as locus coeruleus, paragigantocellular nucleus, spinal cord. Acute and chronic application of opiates and withdrawal give rise to cellular adaptations which depend on the nature and efficacy of the opiate, the type of receptor and second messengers, as well as the type of cell line under study. These cellular mechanisms have consequences on neuronal excitability and gene expression. They constitute a model of cellular tolerance and dependence, but cannot explain the subtelties encountered in clinical practice.
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Narcóticos/farmacología , Trastornos Relacionados con Opioides , Enfermedad Crónica , Tolerancia a Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Narcóticos/efectos adversos , Neuronas/efectos de los fármacos , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Receptores Opioides/efectos de los fármacos , Receptores Opioides/genética , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuroendocrine function, the release of arginine vasopressin (AVP) by the hypothalamo-neurohypophyseal axis. Treatment of mouse-isolated hypothalami and neurointermediate lobes (NIL) with H2O2 increased AVP release. This effect was inhibited by copper-zinc superoxide dismutase-1 (SOD1) analogs. By measuring cGMP accumulation as an indicator of biologically active NO, we found that H2O2 treatment decreased cGMP formation in both hypothalami and NIL. We have previously shown that NO inhibits AVP release by a cGMP-independent mechanism. Given that H2O2 stimulated AVP release, while it reduced cGMP production, our findings strongly suggest that oxidative damage affects neurosecretion by reducing NO availability. To test whether such a mechanism may operate under pathological conditions with pronounced oxidative stress, we compared neurosecretion in wild-type and transgenic mice carrying a mutated form of SOD1 associated with human familial amyotrophic lateral sclerosis. Reminiscent of the data obtained from H2O2-treated tissues, hypothalami and NIL from SOD1 mutants displayed decreased cGMP accumulation and increased AVP release, compared with tissues from wild-type littermates. Since neuronal NO synthase expression was not modified, we conclude that the perturbed free radical metabolism associated with the SOD1 mutation is likely to trap NO, and thereby alter neurosecretion, a mechanism that can be exacerbated in specific physiopathological conditions.
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Esclerosis Amiotrófica Lateral/genética , Sistema Hipotálamo-Hipofisario/metabolismo , Mutación/fisiología , Sistemas Neurosecretores/metabolismo , Estrés Oxidativo/fisiología , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/enzimología , Animales , Western Blotting , AMP Cíclico/metabolismo , GMP Cíclico/biosíntesis , Femenino , Peróxido de Hidrógeno/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/genética , Ratones , Ratones Transgénicos , Neuropéptidos/farmacología , Sistemas Neurosecretores/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo I , Oxidantes/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Superóxido Dismutasa/farmacologíaRESUMEN
We have previously shown that reductions in c-Myb-dependent transcription inhibit cell cycle progression and decrease intracellular Ca2+ concentrations in vascular smooth muscle cells (VSMC). We now report that these effects are largely mediated by a 4- to 10-fold increased rate of La(3+)-sensitive 45Ca extrusion, which is associated with 2- to 4-fold increased levels of plasma membrane Ca(2+)-ATPase 1 (PMCA1) mRNA and protein. PMCA4 mRNA, present at much lower concentrations, undergoes similar changes during suppression of c-Myb activity. We also report that PMCA1 expression is regulated during VSMC cell cycle progression, such that levels of PMCA1 are 40% lower at the G1/S interface than at G0. Moreover, transient overexpression of PMCA1a in VSMC elevates the 45Ca efflux rate by approximately 2-fold, decreases resting and peak thapsigargin-releasable Ca2+ concentrations at G1/S by 43% (68 nM) and 52% (160 nM), respectively, and reduces the rate of cell proliferation by over 2.5-fold. These data define a mechanism for c-Myb-dependent Ca2+ homeostasis and support a critical role for PMCA in the regulation of VSMC growth.
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ATPasas Transportadoras de Calcio/biosíntesis , Calcio/metabolismo , Ciclo Celular/fisiología , Membrana Celular/enzimología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Factores de Transcripción , Animales , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de Transporte de Catión , División Celular/efectos de los fármacos , Línea Celular Transformada , Proteínas de Homeodominio/biosíntesis , Homeostasis , Isoenzimas/biosíntesis , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-myb , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Tapsigargina/farmacología , Transactivadores/biosíntesis , Transactivadores/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Children dying of perinatally-acquired HIV-infection may move from one chaotic system to another as medical and social services scramble to meet the growing need. Incarceration, addiction, illness, abandonment or court removal of children, hospitalization, sibling separation or homelessness may all contribute to family death, even before the physical death of family members. Early planning is essential to prevent further chaos and to develop placement and care strategies which provide for the emotional survival of young children facing physical and familial devastation. Considerations for such planning are discussed, with practical suggestions for supporting the child through change, loss and death.