Departamento de Biología Experimental

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A Comparative Analysis of Mitogenomes in Species of the Tapinoma nigerrimum Complex and Other Species of the Genus Tapinoma (Formicidae, Dolichoderinae)
(MDPI, 2024-12-02) Ruiz-Mena, Areli; Mora, Pablo; Rico-Porras, José M.; Kaufmann, Bernard; Seifert, Bernhard; Palomeque, Teresa; Lorite, Pedro
Using next-generation sequencing data, the complete mitogenomes of six species from the genus Tapinoma were assembled. This study explores the mitochondrial genomes of Tapinoma species, among them the five species from the Tapinoma nigerrimum complex, comparing them with each other and with other species from Dolichoderinae subfamily to understand their evolutionary relationships and evolution. Tapinoma mitochondrial genomes contain the typical set of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and the A + T-rich control region. A phylogenetic analysis using the protein-coding gene sequences from available Dolichoderinae mitogenomes supports the monophyletic nature of the genus Tapinoma, with the T. nigerrimum complex forming a well-supported clade. Key findings include genetic traits unique to the T. nigerrimum complex, such as a start codon in the atp8 gene and a complete stop codon in cox1, distinguishing them from other Tapinoma species. Additionally, a gene rearrangement involving tRNA-Trp, tRNA-Cys, and tRNA-Tyr was found exclusively in the Tapinoma species, suggesting a potential phylogenetic marker for the genus.
A MicroRNA-Transcription Factor Blueprint for Early Atrial Arrhythmogenic Remodeling
(Wiley, 2015-06-28) Torrado, Mario; Franco, Diego; Lozano-Velasco, Estefanía; Hernández-Torres, Francisco; Calviño, Ramón; Aldama, Guillermo; Centeno, Alberto; Castro-Beiras, Alfonso
Spontaneous self-terminating atrial fibrillation (AF) is one of the most common heart rhythm disorders, yet the regulatory molecular mechanisms underlying this syndrome are rather unclear. MicroRNA (miRNA) transcriptome and expression of candidate transcription factors (TFs) with potential roles in arrhythmogenesis, such as Pitx2, Tbx5, and myocardin (Myocd), were analyzed bymicroarray, qRT-PCR, andWestern blotting in left atrial (LA) samples from pigs with transitory AF established by right atrial tachypacing. Induced ectopic tachyarrhythmia caused rapid and substantial miRNA remodeling associated with a marked downregulation of Pitx2, Tbx5, and Myocd expression in atrial myocardium. The downregulation of Pitx2, Tbx5, and Myocd was inversely correlated with upregulation of the corresponding targeting miRNAs (miR-21, miR-10a/10b, and miR-1, resp.) in the LA of paced animals. Through in vitro transient transfections of HL-1 atrial myocytes, we further showed that upregulation of miR-21 did result in downregulation of Pitx2 in cardiomyocyte background. The results suggest that immediate-early miRNA remodeling coupled with deregulation of TF expression underlies the onset of AF.
A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate
(Taylor and Francis, 2015-06-08) Lozano-Velasco, Estefanía; Vallejo-Pulido, Daniel; Esteban-Ruiz, Francisco José; Doherty, Chris; Hernández-Torres, Francisco; Franco, Diego; Aránega, Amelia Eva
The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5 satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine.
Age-related changes of the nitric oxide system in the rat brain
(Elsevier BV, 2002) Siles, Eva; Martínez-Lara, Esther; Cañuelo, Ana; Sánchez, Marta; Hernández-Cobo, Raquel; López-Ramos, Juan Carlos; del-Moral, María Luisa; Esteban-Ruiz, Francisco José; Blanco-Ruiz, Santos; Pedrosa-Raya, Juan ángel; Rodrigo, José; Peinado-Herreros, María Ángeles
This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.
Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases
(OXFORD UNIV PRESS, 2020-01) Matamoros, Manuel Ángel; Cutrona, María C.; Wienkoop, Stefanie; Begara-Morales, Juan Carlos; Sandal, Niels; Orera, Irene; Barroso-Albarracín, Juan Bautista; Stougaard, Jens; Becana, Manuel
Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosyla tion. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cas cades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionyla tion inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to develop mental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in leg ume biology.
Are patients with fibromyalgia in a prothrombotic state?
(Sage Publications Inc, 2019) Molina-Ortega, Francisco Javier; del-Moral, María Luisa; la-Rubia, Mercedes; Blanco-Ruiz, Santos; Carmona-Martos, Ramón; Rus-Martínez, Alma
Objectives: The aim of this study was to investigate thrombosis-related parameters (blood coagulation parameters, platelet indices, red blood cell [RBC] count, and inflammatory markers) in patients with fibromyalgia (FM). Method: We carried out a case–control study with 35 women with FM and 12 age-matched healthy volunteers to analyze fibrinogen levels, prothrombin time, cephaline time, platelet count, platelet distribution width (PDW), mean platelet volume (MPV), RBC count, neutrophil-tolymphocyte ratio, and platelet-to-lymphocyte ratio (PLR). Results: The results showed significantly increased fibrinogen levels (p < .05), platelet count (p < .05), PDW (p ¼ .059), RBC count (p < .05), and PLR (p < .05) in women with FM versus the healthy volunteers. Prothrombin time (p < .05) and MPV (p < .05) were significantly lower in patients with FM than in the controls. Conclusions: Elevated platelet and RBC counts, PDW values, and fibrinogen levels as well as decreased prothrombin time are all indicative of a prothrombotic state in FM patients, which may be enhanced by an increased inflammatory tone. This prothrombotic state may increase the risk of thrombosis-related cardiovascular disease in patients with FM.
Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases
(MDPI, 2022) Peinado-Herreros, María Ángeles; Ovelleiro, David; del-Moral, María Luisa; Hernández-Cobo, Raquel; Martínez-Lara, Esther; Siles, Eva; Pedrajas, José Rafael; García-Martín, María Luisa; Caro, Carlos; Peralta, Sebastián; Morales-Hernández, María Encarnación; Ruiz-Martínez, María Adolfina; Blanco-Ruiz, Santos
Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies.
Celiac Immunogenic Potential of α-Gliadin Epitope Variants from Triticum and Aegilops Species
(MDPI, 2019-01) Ruiz-Carnicer, Ángela; Comino-Montilla, Isabel; Segura, Veronica; Ozuna, Carmen Victoria; Moreno-Amador, María Lourdes; López-Casado, Miguel Ángel; Torres-López, María Isabel; Barro, Francisco; Sousa, Carolina
This study investigates the immunotoxic potential of α-gliadins in wheat species and their implications for celiac disease (CD). Gluten, the primary structural protein complex in wheat, contains α-gliadins, which are rich in T-cell stimulating epitopes (DQ2.5-glia-α1, DQ2.5-glia-α2, and DQ2.5-glia-α3) that trigger immune responses in individuals with CD. The analysis spanned diploid, tetraploid, and hexaploid wheat species, focusing on the abundance and immunostimulatory capacity of canonical epitopes and their variants.The findings reveal that DQ2.5-glia-α1 and DQ2.5-glia-α3 are more prevalent than DQ2.5-glia-α2. Canonical DQ2.5-glia-α1 is notably abundant in genomes of the BBAADD, AA, and DD types, while DQ2.5-glia-α3 epitope variants are highly represented in BBAADD and BBAA wheats, despite a lower presence of the canonical form. Importantly, the introduction of a natural amino acid substitution (Q to H) at any position effectively neutralized the immunotoxicity of the epitopes without compromising wheat's functional properties.This work underscores the potential of targeted amino acid substitutions as a strategy to develop wheat varieties that are safer for individuals with CD while preserving their technological value.
Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components
(Wiley, 2002-10-31) Lalioti, Vasiliki S.; Pérez-Fernández, Jorge; Remacha, Miguel; García Ballesta, Juan Pedro
The interactions among the yeast stalk components(P0, P1⍺, P1ꞵ, P2⍺ and P2ꞵ) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1⍺ and P1ꞵ were found to interact with P2ꞵ and P2⍺ respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1⍺ with P2ꞵ and of P1ꞵ with P2⍺ rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.
Comparative cytogenetics of Lachnaia species (Coleoptera: Chrysomelidae) reveals a novel telomeric motif (TTTGG) in insects
(Springer, 2025-03-27) Rico-Porras, Jose Manuel; Mora-Ruiz, Pablo; Palomeque, Teresa; Cabral-de-Mello, Diogo; Lorite, Pedro
Cytogenetic analyses of Lachnaia hirta, Lachnaia tristigma, and Lachnaia vicina have revealed that all species show a similar karyotype with 2n=24 and a meioformula of 11+Xy+. Despite this, their sex chromosomes show certain diferences in morphology. All species exhibit pericentromeric heterochromatin on all autosomes and X chromosomes, with the Y chromosome being heterochromatic. The X and Y chromosomes carry the nucleolar organizer regions in the three species, althoug L. vicina also has them in one autosomal pair. No clear hybridization signals were obtained using a probe with the TTAGG repeat, which is the ancestral DNA motif of telomeres in insects. The genomes of the three species have been sequenced. The obtained data have been used for the identifcation of telomeric motifs through bioinformatics analyses, including the Telomeric Repeats Identifcation Pipeline, a bioinformatics tool for identifying telomeric repeat motifs. According to the data, the TTTGG sequence is suggested as the telomeric repeat in these species, a fnding confrmed by fuorescence in situ hybridization. The TTTGG motif constitutes a new telomeric repeat motif in Coleoptera. This new motif has been found at the chromosome ends of another beetle species, the oak borer Platypus cylindrus (Coleoptera, Curculionidae), and in a wasp species, Oxytorus armatus (Hymenoptera, Ichneumonoidea), whose genomes are assembled at the chromosome level. The detection of this telomeric repeat in two families of beetles and in a hymenopteran species suggests that this new telomeric motif has independently emerged in evolutionarily distant groups of insects.
Comparative proteomic study of early hypoxic response in the cerebral cortex of rats submitted to two different hypoxic models
(Wiley - V C H Verlag GmbH, 2017) Ovelleiro, David; Blanco-Ruiz, Santos; Hernández-Cobo, Raquel; Peinado-Herreros, María Ángeles
Purpose: The present study analyses and compares the cortical brain proteomic profiles of two different models of cerebral hypoxic insult in rats (HH: hypobaric hypoxia and HHI: ischemia followed by hypobaric hypoxia) in an attempt to describe the alterations of the early molecular hypoxic adaptive response underlying each one. Experimental Design: A quantitative proteomic profile of left-brain cortices of rats under HH, HHI, and control conditions was determined using isobaric labeling (Tandem Mass TagTM) on the protein extracts from pools of five individuals. Data are available via ProteomeXchange with identifier PXD004091. Results: Altogether, 339 proteins were confidently quantified, 99 of them showing significant variations in the hypoxic conditions with respect to the control. The HHI model presents a global effect of protein downregulation while HH produces an overall increase of the protein levels. While HH mainly affecting oxidative and energetic metabolism, HHI also interferes with synaptic transmission, neurotransmitter secretion, substantia nigra development, and triggers apoptosis through mitochondrial pathway. Conclusions and Clinical Relevance: The findings obtained show an overview of protein alterations under two hypoxic models of different aetiology and provide a basis for more detailed studies in order to unravel new specific mechanisms and therapies for hypoxic pathologies.
Complex Evolutionary Histor of Mboumar, a Mariner Element Widely Represented in Ant Genomes
(Nature Research, 2020-02) Sanllorente, Olivia; Vela, Jesús; Mora, Pablo; Ruiz-Mena, Areli; Torres, María Isabel; Lorite, Pedro; Palomeque, Teresa
This study examines the mariner-like transposable element Mboumar, previously identified in the ant Messor bouvieri, across 22 ant species from nine subfamilies, including both primitive and derived lineages. The widespread presence of Mboumar-like elements in ant genomes is evident, but the phylogenetic relationships of these elements do not align with the evolutionary history of their ant hosts. Genetic analysis revealed conserved transposable elements with uninterrupted open reading frames in 11 species, encoding transposases closely resembling the active Mboumar-9 transposase. Selection tests indicate purifying selection has shaped the evolution of these elements.
Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana
(PUBLIC LIBRARY SCIENCE, 2014-10-21) Chaki, Mounira; Kovacs, Izabella; Spannagl, Manuel; Lindermayr, Christian
Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments ‘‘chloroplast,’’ ‘‘CUL4-RING ubiquitin ligase complex,’’ and ‘‘membrane’’ more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.
Constitutive nitric oxide synthases are responsible for the nitric oxide production in the ischemic aged cerebral cortex
(Elsevier BV, 2005) Martínez-Lara, Esther; Cañuelo, Ana; Siles, Eva; Hernández-Cobo, Raquel; del-Moral, María Luisa; Blanco-Ruiz, Santos; Pedrosa-Raya, Juan ángel; Rodrigo, José; Peinado-Herreros, María Ángeles
Aged brain shows reduced biological plasticity to meet emergency conditions such as ischemia, a process in which nitric oxide (NO) and apoptosis have been shown to play important roles. Using a model of transient global ischemia, we have analyzed the NO system and the p53, bax and bcl-2 response in the cerebral cortex of aged rats. Although immediately after ischemia the NO level is maintained, the reperfusion period increases NO concentrations together with the following: (i) greater bulk-protein nitration mainly due to a 50-kDa immunoreactive band; (ii) an increase in p53 protein; and (iii) an up-regulation of Bax together with a down-regulation of Bcl-2. These results match up with induced endothelial nitric oxide synthase expression immediately after ischemia and in neuronal nitric oxide synthase with the reperfusion. However, inducible nitric oxide synthase was not altered with ischemia/reperfusion. Altogether, these data suggest that NO production in cerebral cortex of aged ischemic animals is due to the constitutive NO synthase isoforms. This response is accompanied by the increased expression of pro-apoptotic proteins.
Coupling the endophytic microbiome with the host transcriptome in olive roots
(2021) Luque, Francisco
Cre recombinase microinjection for single-cell tracing and localised gene targeting
(COMPANY BIOLOGISTS LTD, 2023-02-15) Sendra, Miquel; Hourcade, Juan de Dios; Temiño, Susana; Sarabia, Antonio José; Ocaña, Oscar H; Domínguez, Jorge N; Torres, Miguel
Las técnicas de análisis de linajes celulares son esenciales para comprender el desarrollo embrionario. Las microinyecciones de colorantes lipofílicos, la transfección viral y la iontoforesis han sido técnicas clave para mapear el origen de las células progenitoras que forman los diferentes órganos en el embrión de ratón post-implantación. Estas técnicas requieren habilidades avanzadas de manipulación, y solo la técnica de iontoforesis, técnica compleja y de eficiencia limitada, ha sido utilizada para el marcaje de células individuales. En este estudio, hemos optimizado una nueva herramienta para llevar a cabo el trazado de linajes celulares y ablación génica localizada, utilizando microinyección de recombinasa Cre permeable a células (TAT-Cre). En primer lugar, hemos “mapeado” el destino de las células progenitoras indiferenciadas hacia las diferentes cámaras del corazón embrionario. Por otro lado, hemos logrado optimizar las condiciones para, ajustando la dosis de TAT-Cre, lo que permite realizar análisis clonales de los progenitores de mesodermo naciente. Finalmente, la microinyección de TAT-Cre en embriones Mycnflox/flox en el tubo cardíaco primitivo reveló que el gen Mycn desempeña un papel autónomo en la proliferación de cardiomiocitos. Esta herramienta ayudará a los investigadores a identificar las células progenitoras y rutas moleculares involucradas en el desarrollo de órganos, contribuyendo a entender el origen de los defectos congénitos.
Crosstalk between hydroxytyrosol, a major olive oil phenol, and HIF-1 in MCF-7 breast cancer cells
(Nature Research, 2020) Calahorra, Jesús; Martínez-Lara, Esther; Granadino-Roldán, José Manuel; Martí, Juan Manuel; Cañuelo, Ana; Blanco-Ruiz, Santos; Oliver, Francisco Javier; Siles, Eva
Olive oil intake has been linked with a lower incidence of breast cancer. Hypoxic microenvironment in solid tumors, such as breast cancer, is known to play a crucial role in cancer progression and in the failure of anticancer treatments. HIF-1 is the foremost effector in hypoxic response, and given that hydroxytyrosol (HT) is one of the main bioactive compounds in olive oil, in this study we deepen into its modulatory role on HIF-1. Our results in MCF-7 breast cancer cells demonstrate that HT decreases HIF-1α protein, probably by downregulating oxidative stress and by inhibiting the PI3K/Akt/mTOR pathway. Strikingly, the expression of HIF-1 target genes does not show a parallel decrease. Particularly, adrenomedullin and vascular endothelial growth factor are up-regulated by high concentrations of HT even in HIF-1α silenced cells, pointing to HIF-1-independent mechanisms of regulation. In fact, we show, by in silico modelling and transcriptional analysis, that high doses of HT may act as an agonist of the aryl hydrocarbon receptor favoring the induction of these angiogenic genes. In conclusion, we suggest that the effect of HT in a hypoxic environment is largely affected by its concentration and involves both HIF-1 dependent and independent mechanisms.
Differential chamber-specific expression and regulation of long non-coding RNAs during cardiac development
(Elsevier, 2019-10) García-Padilla, Carlos; Domínguez-Macías, Jorge Nicolás; Aránega, Amelia Eva; Franco, Diego
Cardiovascular development is governed by a complex interplay between inducting signals such as Bmps and Fgfs leading to activation of cardiac specific transcription factors such as Nkx2.5, Mef2c and Srf that orchestrate the initial steps of cardiogenesis. Over the last decade we have witnessed the discovery of novel layers of gene regulation, i.e. post-transcriptional regulation exerted by non-coding RNAs. The function role of small non coding RNAs has been widely demonstrated, e.g. miR-1 knockout display several cardiovascular abnormalities during embryogenesis. More recently long non-coding RNAs have been also reported to modulate gene expression and function in the developing heart, as exemplified by the embryonic lethal phenotypes of Fendrr and Braveheart knock out mice, respectively. In this study, we investigated the differential expression profile during cardiogenesis of previously reported lncRNAs in heart development. Our data revealed that Braveheart, Fendrr, Carmen display a preferential adult expression while Miat, Alien, H19 preferentially display chamber-specific expression at embryonic stages. We also demonstrated that these lncRNAs are differentially regulated by Nkx2.5, Srf and Mef2c, Pitx2 > Wnt > miRNA signaling pathway and angiotensin II and thyroid hormone administration. Importantly isoform-specific expression and distinct nuclear vs cytoplasmic localization of Braveheart, Carmen and Fendrr during chamber morphogenesis is observed, suggesting distinct functional roles of these lncRNAs in atrial and ventricular chambers. Furthermore, we demonstrate by in situ hybridization a dynamic epicardial, myocardial and endocardial expression of H19 during cardiac development. Overall our data support novel roles of these lncRNAs in different temporal and tissue-restricted fashion during cardiogenesis.
Differential modulation of S-nitrosoglutathione reductase and reactive nitrogen species in wild and cultivated tomato genotypes during development and powdery mildew infection.
(ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2020-10) Jahnová, Jana; Činčalová, Lucie; Sedlářová, Michaela; Jedelská, Tereza; Sekaninová, Jana; Mieslerová, Barbora; Luhová, Lenka; Barroso-Albarracín, Juan Bautista ; Petřivalský, Marek
Nitric oxide plays an important role in the pathogenesis of Pseudoidium neolycopersici, the causative agent of tomato powdery mildew. S-nitrosoglutathione reductase, the key enzyme of S-nitrosothiol homeostasis, was investigated during plant development and following infection in three genotypes of Solanum spp. differing in their resistance to P. neolycopersici. Levels and localization of reactive nitrogen species (RNS) including NO, S nitrosoglutathione (GSNO) and peroxynitrite were studied together with protein nitration and the activity of nitrate reductase (NR). GSNOR expression profiles and enzyme activities were modulated during plant devel opment and important differences among Solanum spp. genotypes were observed, accompanied by modulation of NO, GSNO, peroxynitrite and nitrated proteins levels. GSNOR was down-regulated in infected plants, with exception of resistant S. habrochaites early after inoculation. Modulations of GSNOR activities in response to pathogen infection were found also on the systemic level in leaves above and below the inoculation site. Infection strongly increased NR activity and gene expression in resistant S. habrochaites in contrast to susceptible S. lycopersicum. Obtained data confirm the key role of GSNOR and modulations of RNS during plant development under normal conditions and point to their involvement in molecular mechanisms of tomato responses to bio trophic pathogens on local and systemic levels.
Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation
(Oxford Academic, 2015-06-25) Begara-Morales, Juan Carlos; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla-Serrano, María Nieves; López-Jaramillo, Javier; Luque-Vázquez, Francisco; Corpas, Francisco Javier; Barroso-Albarracín, Juan Bautista
The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of NO and reactive oxygen species metabolism in antioxidant defence against nitro-oxidative stress situations in plants.

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