RESUMO
The formation and the chemical characterization of single atoms of dubnium (Db, element 105), in the form of its volatile oxychloride, was investigated using the on-line gas phase chromatography technique, in the temperature range 350-600 °C. Under the exactly same chemical conditions, comparative studies with the lighter homologues of Groupâ 5 in the Periodic Table clearly indicate the volatility sequence being NbOCl3 > TaOCl3 ≥ DbOCl3 . From the obtained experimental results, thermochemical data for DbOCl3 were derived. The present study delivers reliable experimental information for theoretical calculations on chemical properties of transactinides.
RESUMO
Somatic KRAS mutations are highly prevalent in many cancers. In addition, a distinct spectrum of germline KRAS mutations causes developmental disorders called RASopathies. The mutant proteins encoded by these germline KRAS mutations are less biochemically and functionally activated than those in cancer. We generated mice harboring conditional KrasLSL-P34Rand KrasLSL-T58I knock-in alleles and characterized the consequences of each mutation in vivo. Embryonic expression of KrasT58I resulted in craniofacial abnormalities reminiscent of those seen in RASopathy disorders, and these mice exhibited hyperplastic growth of multiple organs, modest alterations in cardiac valvulogenesis, myocardial hypertrophy, and myeloproliferation. By contrast, embryonic KrasP34R expression resulted in early perinatal lethality from respiratory failure due to defective lung sacculation, which was associated with aberrant ERK activity in lung epithelial cells. Somatic Mx1-Cre-mediated activation in the hematopoietic compartment showed that KrasP34R and KrasT58I expression had distinct signaling effects, despite causing a similar spectrum of hematologic diseases. These potentially novel strains are robust models for investigating the consequences of expressing endogenous levels of hyperactive K-Ras in different developing and adult tissues, for comparing how oncogenic and germline K-Ras proteins perturb signaling networks and cell fate decisions, and for performing preclinical therapeutic trials.
Assuntos
Cardiomiopatias/patologia , Craniossinostoses/patologia , Doenças Hematológicas/patologia , Pneumopatias/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Craniossinostoses/etiologia , Craniossinostoses/metabolismo , Feminino , Doenças Hematológicas/etiologia , Doenças Hematológicas/metabolismo , Pneumopatias/etiologia , Pneumopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
Tissue regenerative potential displays striking divergence across phylogeny and ontogeny, but the underlying mechanisms remain enigmatic. Loss of mammalian cardiac regenerative potential correlates with cardiomyocyte cell-cycle arrest and polyploidization as well as the development of postnatal endothermy. We reveal that diploid cardiomyocyte abundance across 41 species conforms to Kleiber's law-the ¾-power law scaling of metabolism with bodyweight-and inversely correlates with standard metabolic rate, body temperature, and serum thyroxine level. Inactivation of thyroid hormone signaling reduces mouse cardiomyocyte polyploidization, delays cell-cycle exit, and retains cardiac regenerative potential in adults. Conversely, exogenous thyroid hormones inhibit zebrafish heart regeneration. Thus, our findings suggest that loss of heart regenerative capacity in adult mammals is triggered by increasing thyroid hormones and may be a trade-off for the acquisition of endothermy.
Assuntos
Coração/fisiologia , Miócitos Cardíacos/fisiologia , Poliploidia , Regeneração/fisiologia , Hormônios Tireóideos/fisiologia , Animais , Regulação da Temperatura Corporal , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Diploide , Camundongos , Miócitos Cardíacos/classificação , Filogenia , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/fisiologia , Regeneração/efeitos dos fármacos , Regeneração/genética , Transdução de Sinais , Hormônios Tireóideos/farmacologia , Peixe-ZebraRESUMO
The mechanisms responsible for active DNA demethylation remain elusive in Metazoa. A previous study that utilized zebrafish embryos provided a potent mechanism for active demethylation in which three proteins, AID, MBD4, and GADD45 are involved. We recently found age-dependent DNA hypomethylation in zebrafish, and it prompted us to examine if AID and MBD4 could be involved in the phenomenon. Unexpectedly, however, we found that most of the findings in the previous study were not reproducible. First, the injection of a methylated DNA fragment into zebrafish eggs did not affect either the methylation of genomic DNA, injected methylated DNA itself, or several loci tested or the expression level of aid, which has been shown to play a role in demethylation. Second, aberrant methylation was not observed at certain CpG islands following the injection of antisense morpholino oligonucleotides against aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA lacked a coding region for the methyl-CpG binding domain, which was assumed to be necessary for guidance to target regions. Taken together, we concluded that there is currently no evidence to support the proposed roles of AID and MBD4 in active demethylation in zebrafish embryos.
Assuntos
Citidina Desaminase/metabolismo , DNA Glicosilases/metabolismo , Metilação de DNA/genética , Embrião não Mamífero/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Ilhas de CpG/genética , Citidina Desaminase/genética , DNA Glicosilases/genética , Metilação de DNA/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Fertilização/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genoma , Injeções , Morfolinos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óvulo/efeitos dos fármacos , Óvulo/metabolismo , Estrutura Terciária de Proteína , Transcrição Gênica/efeitos dos fármacos , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown. RESULTS: To investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration. CONCLUSION: Taken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.
Assuntos
Nadadeiras de Animais/fisiologia , Complexos Multiproteicos/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Nadadeiras de Animais/citologia , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Regeneração , Proteínas Quinases S6 Ribossômicas/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/fisiologiaRESUMO
Although dedifferentiation, transformation of differentiated cells into progenitor cells, is a critical step in the regeneration of amphibians and fish, the molecular mechanisms underlying this process, including epigenetic changes, remain unclear. Dot blot assays and immunohistochemical analyses revealed that, during regeneration of zebrafish fin, the levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are transiently reduced in blastema cells and cells adjacent to the amputation plane at 30 h post-amputation (hpa), and the level of 5mC, but not 5hmC, is almost restored by 72 hpa. We observed that the dedifferentiated cells showed reduced levels of 5mC and 5hmC independent of cell proliferation by 24 hpa. Furthermore, expressions of the proposed demethylation- and DNA repair-related genes were detected during fin regeneration. Taken together, our findings illustrate that the transient reduction of 5mC and 5hmC in dedifferentiated cells is associated with active demethylation during regeneration of zebrafish fin.
Assuntos
5-Metilcitosina/metabolismo , Nadadeiras de Animais/fisiologia , Desdiferenciação Celular , Citosina/análogos & derivados , Metilação de DNA , Regeneração/genética , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Nadadeiras de Animais/citologia , Animais , Animais Geneticamente Modificados , Citosina/metabolismo , Reparo do DNA , Embrião não Mamífero , Epigênese Genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Leucine-rich repeat (LRR)-containing G protein-coupled receptors (LGRs) belong to the superfamily of G protein-coupled receptors, and are characterized by the presence of seven transmembrane domains and an extracellular domain that contains a series of LRR motifs. Three Lgr proteins - Lgr4, Lgr5, and Lgr6 - were identified as members of the LGR subfamily. Mouse Lgr4 has been implicated in the formation of various organs through regulation of cell proliferation during development, and Lgr5 and Lgr6 are stem cell markers in the intestine or skin. Although the expression of these three genes has already been characterized in adult mice, their expression profiles during the embryonic and larval development of the organism have not yet been defined. We cloned two zebrafish lgr genes using the zebrafish genomic database. Phylogenetic analyses showed that these two genes are orthologs of mammalian Lgr4 and Lgr6. Zebrafish lgr4 is expressed in the neural plate border, Kupffer's vesicle, neural tube, otic vesicles, midbrain, eyes, forebrain, and brain ventricular zone by 24h post-fertilization (hpf). From 36 to 96hpf, lgr4 expression is detected in the midbrain-hindbrain boundary, otic vesicles, pharyngeal arches, cranial cartilages such as Meckel's cartilages, palatoquadrates, and ceratohyals, cranial cavity, pectoral fin buds, brain ventricular zone, ciliary marginal zone, and digestive organs such as the intestine, liver, and pancreas. In contrast, zebrafish lgr6 is expressed in the notochord, Kupffer's vesicle, the most anterior region of diencephalon, otic vesicles, and the anterior and posterior lateral line primordia by 24hpf. From 48 to 72hpf, lgr6 expression is confined to the anterior and posterior neuromasts, otic vesicles, pharyngeal arches, pectoral fin buds, and cranial cartilages such as Meckel's cartilages, ceratohyals, and trabeculae. Our results provide a basis for future studies aimed at analyzing the functions of zebrafish Lgr4 and Lgr6 in cell differentiation and proliferation during organ development.