RESUMEN
Diatoms are microalgae that live in marine and freshwater environments and are responsible for about 20% of the world's carbon fixation. Population dynamics of these cells is finely regulated by intricate signal transduction systems, in which oxylipins are thought to play a relevant role. These are oxygenated fatty acids whose biosynthesis is initiated by a lipoxygenase enzyme (LOX) and are widely distributed in all phyla, including diatoms. Here, we present a de novo transcriptome obtained from the RNA-seq performed in the diatom species Pseudo-nitzschia arenysensis, using both a wild-type and a LOX-silenced strain, which will represent a reliable reference for comparative analyses within the Pseudo-nitzschia genus and at a broader taxonomic scale. Moreover, the RNA-seq data can be interrogated to go deeper into the oxylipins metabolic pathways.
Asunto(s)
Diatomeas , Lipooxigenasa , Transcriptoma , Diatomeas/genética , Diatomeas/enzimología , Lipooxigenasa/genética , Lipooxigenasa/metabolismo , Oxilipinas/metabolismoRESUMEN
Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.
Asunto(s)
Diatomeas , Diatomeas/metabolismo , Lipooxigenasa/genética , Lipooxigenasa/metabolismo , Oxilipinas/metabolismo , TranscriptomaRESUMEN
Geleophysic dysplasia (GPHYSD1, MIM231050; GPHYSD2, MIM614185; GPHYSD3, MIM617809) is an autosomal disorder characterized by short-limb dwarfism, brachydactyly, cardiac valvular disease, and laryngotracheal stenosis. Mutations in ADAMTSL2, FBN1, and LTBP3 genes are responsible for this condition. We found that three previously described cases of GPHYSD diagnosed clinically were homozygote or compound heterozygotes for five ADAMTSL2 variants, four of which not being previously reported. By electron microscopy, skin fibroblasts available in one case homozygote for an ADAMTSL2 variant showed a defective intracellular localization of mutant ADAMTSL2 protein that did not accumulate within lysosome-like intra-cytoplasmic inclusions. Moreover, this mutant ADAMTSL2 protein was less secreted in medium and resulted in increased SMAD2 phosphorylation in transfected HEK293 cells.
RESUMEN
BACKGROUND: Geleophysic dysplasia (GPHYSD) is a disorder characterized by dysmorphic features, stiff joints and cardiac involvement due to defects of TGF-ß signaling. GPHYSD can be caused by mutations in FBN1, ADAMTLS2, and LTBP3 genes. METHODS AND RESULTS: Consistent with previous reports, we found intracellular inclusions of unknown material by electron microscopy (EM) in skin fibroblasts of two GPHYSD individuals carrying FBN1 mutations. Moreover, we found that the storage material is enclosed within lysosomes and is associated with the upregulation of several lysosomal genes. Treatment of GPHYSD fibroblasts carrying FBN1 mutations with the angiotensin II receptor type 1 inhibitor losartan that inhibits TGF-ß signaling did not reduce the storage but improved the extracellular deposition of fibrillin-1 microfibrils. CONCLUSION: Losartan is a promising candidate drug for treatment of GPHYSD due to FBN1 defects.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/metabolismo , Fibrilina-1/genética , Fibroblastos/metabolismo , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/metabolismo , Losartán/farmacología , Lisosomas/metabolismo , Microfibrillas/metabolismo , Piel/metabolismo , Piel/patología , Adolescente , Enfermedades del Desarrollo Óseo/patología , Niño , Preescolar , Matriz Extracelular , Femenino , Fibroblastos/ultraestructura , Humanos , Lactante , Deformidades Congénitas de las Extremidades/patología , Masculino , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Diatoms and copepods are main actors in marine food webs. The prey-predator interactions between them affect bloom dynamics, shape marine ecosystems and impact the energy transfer to higher trophic levels. Recently it has been demonstrated that the presence of grazers may affect the diatom prey beyond the direct effect of grazing. Here, we investigated the response of the chain-forming centric diatom Skeletonema marinoi to grazer cues, including changes in morphology, gene expression and metabolic profile. S. marinoi cells were incubated with Calanus finmarchicus or with Centropages typicus and in both cases responded by reducing the chain length, whereas changes in gene expression indicated an activation of stress response, changes in the lipid and nitrogen metabolism, in cell cycle regulation and in frustule formation. Transcripts linked to G protein-coupled receptors and to nitric oxide synthesis were differentially expressed suggesting involvement of these signalling transduction pathways in the response. Downregulation of a lipoxygenase in the transcriptomic data and of its products in the metabolomic data also indicate an involvement of oxylipins. Our data contribute to a better understanding of the gene function in diatoms, providing information on the nature of genes implicated in the interaction with grazers, a crucial process in marine ecosystems.
Asunto(s)
Copépodos/metabolismo , Diatomeas/metabolismo , Transcriptoma , Animales , Ciclo Celular , Regulación hacia Abajo , Ecosistema , Cadena Alimentaria , Perfilación de la Expresión Génica , Metabolismo de los Lípidos , Lípidos/química , Metaboloma , Nitrógeno/química , Oxilipinas/metabolismo , Fenotipo , Filogenia , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
We report the genetic transformation of the planktonic diatoms Pseudo-nitzschia arenysensis and Pseudo-nitzschia multistriata, members of the widely distributed and ecologically important genus Pseudo-nitzschia. P. arenysensis and P. multistriata present the classical size reduction/restitution life cycle and can reproduce sexually. Genetic transformation was achieved with the biolistic method, using the H4 gene promoter from P. multistriata to drive expression of exogenous genes. The transformation was first optimized introducing the Sh ble gene to confer resistance to the antibiotic zeocin. Integration of the transgene was confirmed by PCR and Southern blot analyses. Subsequently, we simultaneously transformed in P. arenysensis two plasmids, one encoding the ß-glucuronidase (GUS) gene together with the plasmid carrying the Sh ble resistance gene, demonstrating the possibility of co-transformation. By transforming a gene encoding a fusion between the histone H4 and the green fluorescent protein (GFP), we demonstrated that fluorescent tagging is possible and that studies for protein localization are feasible. Importantly, we crossed P. arenysensis- and P. multistriata-transformed strains with a wild-type strain of opposite mating type and demonstrated that the transgene can be inherited in the F1 generation. The possibility to transform two diatom species for which genetic crosses are possible opens the way to a number of new approaches, including classical loss of function screens and the possibility to obtain different combinations of double transformants.
Asunto(s)
Biolística/métodos , Diatomeas/genética , Transformación Genética/genética , Transgenes/genética , Bleomicina , Southern Blotting , Cruzamientos Genéticos , Cartilla de ADN/genética , Diatomeas/fisiología , Farmacorresistencia Microbiana/genética , Proteínas Fluorescentes Verdes/genética , Histonas/genética , Patrón de Herencia/genética , Microscopía Fluorescente , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Reproducción/genética , Reproducción/fisiología , Especificidad de la Especie , Transformación Genética/fisiologíaRESUMEN
Myhre syndrome (MS, MIM 139210) is a connective tissue disorder that presents with short stature, short hands and feet, facial dysmorphic features, muscle hypertrophy, thickened skin, and deafness. Recurrent missense mutations in SMAD4 encoding for a transducer mediating transforming growth factor ß (TGF-ß) signaling are responsible for MS. We found that MS fibroblasts showed increased SMAD4 protein levels, impaired matrix deposition, and altered expression of genes encoding matrix metalloproteinases and related inhibitors. Increased TGF-ß signaling and progression of aortic root dilation in Marfan syndrome can be prevented by the antihypertensive drug losartan, a TGF-ß antagonists and angiotensin-II type 1 receptor blocker. Herein, we showed that losartan normalizes metalloproteinase and related inhibitor transcript levels and corrects the extracellular matrix deposition defect in fibroblasts from MS patients. The results of this study may pave the way toward therapeutic applications of losartan in MS.