42 research outputs found
Duplicación, organización genómica y regulación transcripcional de los receptores de la hormona del crecimiento de peces. Aspectos básicos y aplicados en dorada Sparus aurata.
Los primeros receptores de hormona de crecimiento (GHR) descritos en peces se agruparon en dos grupos, las secuencias descritas en peces salmónidos y las descritas en peces no salmónidos. Las de peces no salmónidos presentaron una gran conservación estructural con las secuencias de mamíferos, pero las secuencias de peces salmónidos muestraron un bajo nivel de identidad aminoacídica con ausencia de un par de cisteínas extracelulares y un diferente patrón de tirosinas intracelulares. Llegados a este punto, se planteó la hipótesis de la existencia de dos formas alternativas para el gen GHR en el genoma de los peces: la forma de los peces no salmónidos (GHR-I) y la de los peces salmónidos (GHR-II). A la luz de esta hipótesis se plantearon los siguientes objetivos: 1) Abordar la caracterización molecular y organización genómica de los receptores de la hormona del crecimiento en peces teleósteos, 2) analizar la divergencia evolutiva de los receptores de la hormona del crecimiento en los peces teleósteos, 3) analizar en dorada el patrón de regulación transcripcional de los receptores de la hormona del crecimiento y 4) definir en dorada el uso de los receptores de la hormona del crecimiento como biomarcadores de crecimiento y estrés por confinamiento y exposición a patógeno.
Para analizar la divergencia evolutiva, se diseñaron cebadores degenerados en base a las secuencias de los GHR descritos previamente en los peces salmónidos. Utilizando estos cebadores en una estrategia de RT-PCR, se determinó la existencia del GHR-II en dorada, trucha y lubina. Las nuevas secuencias de GHR-II presentaron las mismas características estructurales que las secuencias de salmónidos y se agruparon con ellas al realizar un análisis filogenético. De esta manera se definió lo que sería en grupo de secuencias de GHR-II de peces mientras que el resto de secuencias de peces corresponderían al GHR-I.
La caracterización molecular de ambos GHR en dorada mostró la misma organización genómica que el GHR de mamíferos, con la salvedad de la falta de un exón homólogo al 3 de mamíferos y la presencia de un intrón adicional en el exón 10 del GHR-I. Además se determinó que ambos receptores presentaron un único sitio de inicio de la transcripción en contraposición a la situación de mamíferos: un único receptor con diferentes inicios de la transcripción. Esta diferente organización sugirió que el gen del GHR se ajusta al modelo de evolución por subfuncionalización.
Para analizar su regulación transcripcional, se midió la expresión de ambos GHR junto con la de IGF-I e IGF-II bajo diferentes modelos experimentales en dorada. Los modelos estudiados fueron ayuno, estación, edad, infección por patógeno (Enteromyxum leei) y estrés por confinamiento. De estas experiencias se dedujo que la mayor de expresión de ambos GHR se dio en hígado y que los cambios en la concentración de IGF-I circulante reflejaron su expresión a nivel hepático. La expresión del GHR-I estuvo altamente correlacionada con la expresión de IGF-I e IGF-II en las experiencias de estación, edad e infección por patógeno tanto a nivel hepático como extrahepático. Sin embargo, en el modelo de estrés por confinamiento, la disminución de la expresión hepática de la IGF-I e IGF-II reflejó los cambios en la expresión del GHR-II. Esto estuvo en consonancia con los sitios de unión de factores de transcripción presentes en su región flanqueante. El modelo propuesto mostró una regulación diferencial de los GHR de dorada, aunque no se pudo excluir un cierto solapamiento funcional con independencia de que fueran promiscuos o específicos de la GH.The first fish growth hormone receptor (GHR) sequences were grouped in two major groups: salmonid fish and non-salmonid fish. Sequences of non-salmonid fish showed a high structural conservation with those of mammals. On the other hand, sequences of salmonid fish showed a low level of aminoacid identity with the lack of two extracellular cysteines and a different intracellular tyrosine pattern. At that moment, the existence of two alternative copies of the GHR gene in fish genomes, GHR-I (non-salmonid form) and GHR-II (salmonid form), was hypothesised. Following that hypothesis, we formulated the following objectives: 1) Address the molecular characterization and genomic organization of GHRs in teleost fish, 2) analyse their evolutional divergence, 3) analyse their transcriptional regulation in gilthead sea bream and 4) define their use in gilthead sea bream as biomarkers of growth and stress.
The presence of GHR-II was demonstrated for the first time in gilthead sea bream, European sea bass and rainbow trout. In addition, the genomic organization of both GHR was elucidated in gilthead sea bream. Fish GHR showed the same genomic organization than human GHR except for the lack of the exon 3 and the presence of a short intron within GHR-I exon 10. Moreover, fish GHRs only contained a transcription start site whereas tetrapods’ GHR has several sites.
The transcriptional regulation of gilthead sea bream GHR-I, GHR-II, IGF-I and IGF-II was assessed in basis to fasting, season, age, stress and parasite challenge (Enteromixum leei). In basis to our results, the expression of GHR-I was higher in hepatic tissue. Moreover, changes in circulating IGF-I levels reflected the IGF-I hepatic expression. GHR-I expression was highly correlated with IGF expression in season, age and parasite challenge at hepatic and extrahepatic level. However, changes in IGF expression under stress were correlated to hepatic GHR-II expression. This fact agreed with the transcription factor binding sites found in the 5’flanquing region of GHR-II. In summary, coexpression analyses suggest a key role of GHR-I in the tissue-specific regulation of IGFs in gilthead sea bream. Some functional redundancy of GHR-I and GHR-II cannot be excluded, emerging GHR-II as a stress and redox-sensitive gene
Automated Scalable Heat Shock Modification for Standard Aquatic Housing Systems
Heat shock is a common technique for inducible gene expression system in a variety of organisms. Heat shock treatment of adult zebrafish is more involved and generally consists of manually transferring fish between housing rack tanks and preheated water tanks or the use of timed heaters in stand-alone aquaria. To avoid excessive fish handling and to take advantage of the continuous flow of a standard housing rack, proposed modifications consisted of installing an aquarium heater inside each tank, manually setting the heater to reach heat shocking temperatures (>37°C) and, after that, testing that every tank responded equally. To address the limitations in the existing systems, we developed a novel modification of standard zebrafish housing racks to perform heat shock treatment in conditions of continuous water flow. By adding an extra manifold to the housing rack and connecting it to a recirculating bath to create a parallel water flow system, we can increase the temperature from standard conditions (28.5°C) to heat shock conditions with high precision (38.0?38.3°C, mean±SD=38.1°C±0.14°C) and minimal variation among experimental tanks (coefficient of variation [CV]=0.04%). This means that there is virtually no need for laborious pretreatment calibrations or continuous adjustments to minimize intertank variation. To test the effectiveness of our design, we utilized this system to induce enhanced green fluorescent protein (EGFP) expression in hsp70-EGFP fish and performed a fin regeneration experiment with hsp70l:dnfgfr1-EGFP fish to confirm that heat-induced gene expression reached physiological levels. In summary, our newly described aquatic heat shock system minimizes effort during heat shock experiments, while ensuring the best water quality and fish welfare and facilitating large heat shock settings or the use of multiple transgenic lines for both research and teaching experiments.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140304/1/zeb.2015.1087.pd
Agouti overexpression in a transgenic model regulates integrity, permeability and electrogenic amino acid transport in zebrafish intestine
Overexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing food intake levels and linear growth. A higher feed intake is unnecessary in transgenic fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. To test this hypothesis, wide scope transcriptomic techniques were used to elucidate the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity. In addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic analysis reveal that amino acid, monocarboxylates, ionic and vitamin transmembrane transporters were substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting augmented intestinal laxity that may enhance paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membraned tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was also enhanced in transgenic animals providing strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency suggests that the melanocortin system, specifically asip1 overexpression, is a potential target for the development of genetically engineered fish displaying improved performance and no differential lipid accumulation.info:eu-repo/semantics/publishedVersio
Anti-TNF Therapies Suppress Adipose Tissue Inflammation in Crohn's Disease
Anti-TNF biologics have been shown to markedly improve the quality of life for patients with Crohn's disease (CD), yet one-third of patients fail to benefit from this treatment. Patients with CD develop a characteristic wrapping of visceral adipose tissue (VAT) in the inflamed intestinal area, termed creeping fat, and it is known that adipose tissue expansion influences the efficacy of anti-TNF drugs. We questioned whether anti-TNF therapies impact the creeping fat in CD, which might affect the outcome of the disease. Adipose tissue biopsies were obtained from a cohort of 14 patients with CD that received anti-TNF drugs and from 29 non-anti-TNF-treated patients (control group) matched by sex, age, and body mass index undergoing surgical interventions for symptomatic complications. We found that anti-TNF therapies restored adipose tissue morphology and suppressed immune cell infiltration in the creeping fat. Additionally, anti-TNF treatments appeared to markedly improve the pro-inflammatory phenotype of adipose-tissue macrophages and adipose-tissue-derived stem cells. Our study provides evidence that anti-TNF medications influence immune cells and progenitor cells in the creeping of patients with CD, suppressing inflammation. We propose that perilesional VAT should be considered when administering anti-TNF therapy in patients with C
Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism
The histone demethylase KDM1A is a multi- faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A(-/-) hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a(-/-) hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells
LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response
Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells
Aquamax: Sustainable aquafeeds to maximize the health effects of farmed fish for consumers
Jornada de Divulgación Proyecto Europeo AQUAMAX celebrada en el Instituto de Acuicultura de Torre de la Sal (Castellón) el 24 de noviembre de 2010.El Instituto de Acuicultura Torre de la Sal del CSIC organiza una jornada que tiene como objetivo divulgar la investigación sobre el proyecto europeo AQUAMAX. Este proyecto centra su investigación en reemplazar en lo posible los piensos basados en pescado y el aceite de pescado usado actualmente para alimentar a los peces por recursos alternativos libres de contaminantes. Se conseguiría así maximizar el crecimiento, mejorar la conversión del alimento e incrementar la salud y el bienestar de los peces criados en piscifactorías. Además aumentaría la seguridad y calidad del producto que llega finalmente a los consumidores.
La jornada contará con la participación del Doctor S. Kaushik del Instituto Nacional para la Investigación Agronómica de Francia, el Doctor Marc Berntssen del Instituto Nacional de Nutrición e Investigación del Alimento Marino de Noruega y el Profesor Jaume Pérez del Instituto de Acuicultura Torre de la Sal del CSIC.
En las diferentes intervenciones se tratarán temas del proyecto AQUAMAX como la calidad de los alimentos, la seguridad en la alimentación, así como diversas investigaciones basadas en las doradas.Peer Reviewe
Dynamics of liver GH/IGF axis and selected stress markers in juvenile gilthead sea bream (Sparus aurata) exposed to acute confinement. Differential stress response of growth hormone receptors
32 p., 1 tabla, 5 figuras y bibliografíaThe time courses of liver GH/IGF axis and selected stress markers were analyzed in juvenile gilthead sea bream (Sparus aurata) sampled at zero time and at fixed intervals (1.5, 3, 6, 24, 72 and 120 h) after acute confinement (120 kg/m3). Fish remained unfed throughout the course of the confinement study, and the fasting-induced increases in plasma growth hormone (GH) levels were partially masked by the GH-stress inhibitory tone. Hepatic mRNA levels of growth hormone receptor-I (GHR-I) were not significantly altered by confinement, but a persistent 2-fold decrease in GHR-II transcripts was found at 24 and 120 h. A consistent decrease in circulating levels of insulin-like growth factor-I (IGF-I) was also found through most of the experimental period, and the down-regulated expression of GHR-II was positively correlated with changes in hepatic IGF-I and IGF-II transcripts. This stress-specific response was concurrent with plasma increases in cortisol and glucose levels, reflecting the cortisol peak (60-70 ng/mL), the intensity and duration of the stressor when data found in the literature were compared. Adaptive responses against oxidative damage were also found, and a rapid enhanced expression was reported in the liver tissue for mitochondrial heat-shock proteins (glucose regulated protein 75). At the same time, the down-regulated expression of proinflammatory cytokines (tumour necrosis factor-α) and detoxifying enzymes (cytochrome P450 1A1) might dictate the hepatic depletion of potential sources of reactive oxygen species. These results provide suitable evidence for a functional partitioning of hepatic GHRs under states of reduced IGF production and changing cellular environment resulting from acute confinement.This work was funded by EU (contract no. SSP98-CT-2004-513692; Combined genetic and functional genomic approaches for stress and disease resistance markers assisted selection in fish and shellfish, AQUAFIRST) and Spanish (Ingenio-2010 Programme; Improvement of Aquaculture Production by the use of biotechnological tools, AQUAGENOMICS) projects. AS-V was recipient of a Spanish PhD fellowship from the Diputación Provincial de Castellón.Peer reviewe
TNF-induced lipolysis in fish adipocytes
26 p., 4 figures, 2 tables and bibliograghyThe present study aimed to analyze adiposity heterogeneity and the role of liver X receptor (LXRα) and peroxisome proliferator-activated receptors (PPARs) as targets of tumour necrosis factor-α (TNFα) in gilthead sea bream (Sparus aurata L.). The screening of twenty fish at the beginning of the warm season identified two major groups with fat and lean phenotypes. Fat fish showed increased liver and mesenteric fat depots. This increased adiposity was concurrent in the adipose tissue to enhanced expression of lipoprotein lipase (LPL), whereas mRNA levels of the hormone sensitive lipase (HSL) remained almost unchanged. The resulting LPL/HSL ratio was thereby highest in fat fish, which suggests that this group of fish has not reached their peak of fat storage capacity. This is not surprising given the increased expression of PPARγ in the absence of a counter-regulatory raise of TNFα. However, this lipolytic cytokine exerted dual effects in primary adipocyte cultures that differ within and between lean and fat fish. One set of fat fish did not respond to TNFα treatment, whereas a second set exhibited a lipolytic response (increased glycerol release) which was apparently mediated by the down-regulated expression of PPARβ. In lean fish, TNFα exerted a strong and non-transcriptionally mediated lipolytic action. Alternatively, TNFα would inhibit lipid deposition via the down-regulated expression of adipogenic nuclear factors(PPARγ and LXRα). TNFα targets are therefore different in fish with lean and fat phenotypes, which is indicative of the complex network involved in the regulation of fish lipid metabolism.This work was funded by EU (FOOD-CT-2006-16249; Sustainable Aquafeeds to Maximise the Health Benefits of Farmed Fish for Consumers, AQUAMAX), and Spanish (CONSOLIDER-INGENIO 2010: Improvement of aquaculture production by the use of biotechnological tools) projects. AS-V was the recipient of a Spanish PhD fellowship from the Diputación Provincial de Castellón. LC was funded by a PhD fellowship (BES-2005-9566) from the Ministerio de Educación y Ciencia (project
AGL-2004-06319-C02-02)