RESUMO
Chelates of Gadolinium (Gd), a lanthanide metal, are employed as contrast agents for magnetic resonance imaging and are released into the aquatic environment where they are an emerging contaminant. We studied the effects of environmentally relevant Gd concentrations on the development of two phylogenetically and geographically distant sea urchin species: the Mediterranean Paracentrotus lividus and the Australian Heliocidaris tuberculata. We found a general delay of embryo development at 24h post-fertilization, and a strong inhibition of skeleton growth at 48h. Total Gd and Ca content in the larvae showed a time- and concentration-dependent increase in Gd, in parallel with a reduction in Ca. To investigate the impact of Gd on the expression of genes involved in the regulation of skeletogenesis, we performed comparative RT-PCR analysis and found a misregulation of several genes involved in the skeletogenic and left-right axis specification gene regulatory networks. Species-specific differences in the biomineralization response were evident, likely due to differences in the skeletal framework of the larvae and the amount of biomineral produced. Our results highlight the hazard of Gd for marine organisms.
Assuntos
Anthocidaris/efeitos dos fármacos , Cálcio/metabolismo , Gadolínio/toxicidade , Paracentrotus/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Anthocidaris/classificação , Anthocidaris/crescimento & desenvolvimento , Desenvolvimento Embrionário/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Paracentrotus/classificação , Paracentrotus/crescimento & desenvolvimento , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization.
Assuntos
Metalotioneína/genética , Família Multigênica , Paracentrotus/classificação , Paracentrotus/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Desenvolvimento Embrionário/genética , Éxons , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ordem dos Genes , Metalotioneína/química , Metais/farmacologia , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5 kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus.
Assuntos
Anidrases Carbônicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Paracentrotus/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Embrião não Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , Ponto Isoelétrico , Peso Molecular , Fases de Leitura Aberta , Especificidade de Órgãos , Paracentrotus/classificação , Paracentrotus/embriologia , Paracentrotus/metabolismo , Filogenia , Domínios Proteicos , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The urchin Paracentrotus lividus has been characterized via previous capture and enhancement of low-abundance proteins with combinatorial peptide ligand libraries (CPLL, ProteoMiner). Whereas in the control only 26 unique gene products could be identified, 82 species could be detected after CPLL treatment. Due to the overwhelming presence of two major proteins-the toposome (a highly glycosylated, modified calcium-binding, iron-less transferrin) and the major yolk proteins, belonging to the class of cell adhesion proteins-which constituted about 70% of the proteome of this biological fluid and strongly interfered with the capture of the minority proteome, no additional proteins could be detected. Yet, at present, this constitutes the most thorough investigation of the proteome of this biological fluid.
