Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.696
Filtrar
1.
Nat Commun ; 11(1): 5357, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097721

RESUMO

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the ß-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2-SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Transcriptoma , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , beta Catenina/metabolismo
2.
Nat Commun ; 11(1): 5369, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097732

RESUMO

GABAA receptors mediate most inhibitory synaptic transmission in the brain of vertebrates. Following GABA binding and fast activation, these receptors undergo a slower desensitization, the conformational pathway of which remains largely elusive. To explore the mechanism of desensitization, we used concatemeric α1ß2γ2 GABAA receptors to selectively introduce gain-of-desensitization mutations one subunit at a time. A library of twenty-six mutant combinations was generated and their bi-exponential macroscopic desensitization rates measured. Introducing mutations at the different subunits shows a strongly asymmetric pattern with a key contribution of the γ2 subunit, and combining mutations results in marked synergistic effects indicating a non-concerted mechanism. Kinetic modelling indeed suggests a pathway where subunits move independently, the desensitization of two subunits being required to occlude the pore. Our work thus hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.


Assuntos
Sistema Nervoso/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Transmissão Sináptica/fisiologia , Animais , Simulação por Computador , Canais Iônicos , Transporte de Íons , Cinética , Modelos Moleculares , Conformação Molecular , Mutação , Oócitos , Conformação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/genética , Xenopus laevis/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(35): 21740-21746, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817533

RESUMO

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel is essential for epithelial salt-water balance. CFTR mutations cause cystic fibrosis, a lethal incurable disease. In cells CFTR is activated through the cAMP signaling pathway, overstimulation of which during cholera leads to CFTR-mediated intestinal salt-water loss. Channel activation is achieved by phosphorylation of its regulatory (R) domain by cAMP-dependent protein kinase catalytic subunit (PKA). Here we show using two independent approaches--an ATP analog that can drive CFTR channel gating but is unsuitable for phosphotransfer by PKA, and CFTR mutants lacking phosphorylatable serines--that PKA efficiently opens CFTR channels through simple binding, under conditions that preclude phosphorylation. Unlike when phosphorylation happens, CFTR activation by PKA binding is completely reversible. Thus, PKA binding promotes release of the unphosphorylated R domain from its inhibitory position, causing full channel activation, whereas phosphorylation serves only to maintain channel activity beyond termination of the PKA signal. The results suggest two levels of CFTR regulation in cells: irreversible through phosphorylation, and reversible through R-domain binding to PKA--and possibly also to other members of a large network of proteins known to interact with the channel.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ânions/metabolismo , Fenômenos Biofísicos , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Ativação do Canal Iônico/fisiologia , Mutagênese Sítio-Dirigida , Nucleotídeos/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp/métodos , Fosforilação , Ligação Proteica/fisiologia , Serina/metabolismo , Canais de Ânion Dependentes de Voltagem/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
4.
Chemosphere ; 259: 127415, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32603964

RESUMO

Amphibians are the most endangered class of vertebrates. In this study, Xenopus laevis frogs were exposed to 0, 1 and 10 mg/L of triadimefon or triadimenol. After 14 or 28 days of exposure, high levels of triadimefon or triadimenol obstructed the growth of frogs. However, low levels of triadimefon induced the growth of females after the longer period of exposure. We also found that the antioxidant enzyme activity and LDH levels in males were higher than those in females after 28-days exposure. In histopathology tests, triadimenol exerted more effect on the ovary while triadimefon exerted more effect on the testes. Additionally, the levels of Estradiol in all 14-day treatments, except 1 mg/L triadimenol, were significantly decreased, however, there was no difference in testosterone levels. Furthermore, triadimefon and triadimenol disrupted the expression of genes controlling hormone homeostasis and reproduction, and this effect depended on the exposure time and the gender of the organism. Our experiments explored the effect of triadimefon and its metabolite on the gonads of frogs and highlighted the role that pesticides are likely to play in the global decline of amphibians.


Assuntos
Fungicidas Industriais/toxicidade , Gônadas/crescimento & desenvolvimento , Triazóis/toxicidade , Xenopus laevis/fisiologia , Animais , Feminino , Fungicidas Industriais/metabolismo , Gônadas/anatomia & histologia , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Masculino , Xenopus laevis/metabolismo
5.
Mol Pharmacol ; 98(4): 292-302, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32690627

RESUMO

Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) and the α7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMP-TQS), previously published as a "silent allosteric modulator" and an antagonist of α7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type α7 and the nonorthosterically activatible C190A α7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMP-TQS proved to be an α7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of α7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive α4ß2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with α7T106, and allosteric activation of α7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity. SIGNIFICANCE STATEMENT: Many synthetic ligands are in use as racemic preparations. We show that one enantiomer of the TQS analog Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, originally reported to lack activity when used as a racemic preparation, is an α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM). The other enantiomer is not a PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons identify essential elements of both the allosteric ligands and receptor binding sites important for these allosteric activities.


Assuntos
Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Xenopus laevis/genética , Receptor Nicotínico de Acetilcolina alfa7/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutação , Estereoisomerismo , Sulfonamidas/química , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética
6.
PLoS Biol ; 18(7): e3000750, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667916

RESUMO

Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation-of the order of 10 pN-applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods' phototransduction.


Assuntos
Transdução de Sinal Luminoso , Mecanotransdução Celular , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Xenopus laevis/metabolismo , Animais , Cálcio/metabolismo , Fluorescência , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Mecanotransdução Celular/efeitos da radiação , Família Multigênica , Estimulação Luminosa , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Canais de Cátion TRPC/genética , Proteínas de Xenopus/genética
7.
Proc Natl Acad Sci U S A ; 117(26): 15075-15084, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32532919

RESUMO

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Cromatina/genética , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Ligação Proteica , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo
8.
Nat Cell Biol ; 22(7): 803-814, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572169

RESUMO

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.


Assuntos
Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Forminas/metabolismo , Melanoma/patologia , Proteínas Musculares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Blástula/citologia , Blástula/metabolismo , Forma Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Forminas/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas Musculares/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
9.
Am J Physiol Cell Physiol ; 319(2): C371-C380, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579473

RESUMO

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.


Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
10.
J Vis Exp ; (157)2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32281977

RESUMO

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.


Assuntos
Colesterol/metabolismo , Mamíferos/metabolismo , Oócitos/metabolismo , Xenopus laevis/metabolismo , Animais , Artérias Cerebrais/metabolismo , Humanos , Lipossomos , Fosfolipídeos/metabolismo , Canais de Potássio/metabolismo , Ratos Sprague-Dawley , beta-Ciclodextrinas/metabolismo
11.
Nat Commun ; 11(1): 1345, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165637

RESUMO

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Animais , Blástula/embriologia , Blástula/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade , Histonas/química , Histonas/genética , Domínios Proteicos , Origem de Replicação , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
12.
J Vis Exp ; (156)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32150168

RESUMO

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.


Assuntos
Cálcio/metabolismo , Hibridização in Situ Fluorescente/métodos , Imagem Molecular/métodos , Neurogênese , Neurônios/citologia , Células-Tronco/citologia , Xenopus laevis/crescimento & desenvolvimento , Animais , Neurônios/metabolismo , Células-Tronco/metabolismo , Xenopus laevis/metabolismo
13.
Nat Commun ; 11(1): 1256, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152320

RESUMO

Vertebrates exhibit specific requirements for replicative H3 and non-replicative H3.3 variants during development. To disentangle whether this involves distinct modes of deposition or unique functions once incorporated into chromatin, we combined studies in Xenopus early development with chromatin assays. Here we investigate the extent to which H3.3 mutated at residues that differ from H3.2 rescue developmental defects caused by H3.3 depletion. Regardless of the deposition pathway, only variants at residue 31-a serine that can become phosphorylated-failed to rescue endogenous H3.3 depletion. Although an alanine substitution fails to rescue H3.3 depletion, a phospho-mimic aspartate residue at position 31 rescues H3.3 function. To explore mechanisms involving H3.3 S31 phosphorylation, we identified factors attracted or repulsed by the presence of aspartate at position 31, along with modifications on neighboring residues. We propose that serine 31-phosphorylated H3.3 acts as a signaling module that stimulates the acetylation of K27, providing a chromatin state permissive to the embryonic development program.


Assuntos
Gastrulação/fisiologia , Histonas/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Cromatina , Histonas/genética , Fosforilação , Análise de Sequência de Proteína , Serina
14.
Zoolog Sci ; 37(1): 61-69, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32068375

RESUMO

Iron is an essential element for hemoglobin synthesis during erythropoiesis. Iron overload, in contrast, adversely affects erythropoiesis and causes organ dysfunction. Research using various animal models may help to elucidate pathophysiological mechanisms induced by excess iron. In the present study, we evaluated the relationship between iron metabolism and erythropoietic activity in the African clawed frog, Xenopus laevis. In X. laevis, both erythropoiesis and iron metabolism occur in the liver. First, we developed a method to quantify iron levels in the liver and plasma using 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl) amino] phenol (Nitroso-PSAP). We then measured iron levels and analyzed hematopoietic parameters in frogs that were orally administered sodium ferrous citrate (SFC). The hepatic iron level increased in the SFC group, but the number of erythrocytes, hematocrit, and hemoglobin concentration did not change, suggesting that the regulation of the production and release of mature erythrocytes in the liver was not directly affected by dietary iron. At four days after administration of 2 mg/kg SFC, the number of immature erythrocytes decreased in the liver. Interestingly, atypical blood cells with hyper-segmented nuclei were observed, identified by acridine orange cell staining; these atypical blood cells were hardly detectable under the steady state. Compared with previously reported results in mice, the increase in the hepatic iron levels was small, but our results indicate that SFC affects hematopoietic activity. These results establish a novel model for iron metabolism and provide new insights into the relationship between iron metabolism and erythropoiesis in vertebrates.


Assuntos
Eritropoese/fisiologia , Sobrecarga de Ferro/fisiopatologia , Fígado/fisiopatologia , Xenopus laevis/fisiologia , Animais , Eritropoese/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Ferro/análise , Ferro/sangue , Ferro/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Modelos Animais , Xenopus laevis/metabolismo
15.
Gen Comp Endocrinol ; 292: 113441, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32084349

RESUMO

In the intestine during metamorphosis of the frog Xenopus laevis, most of the larval epithelial cells are induced to undergo apoptosis by thyroid hormone (TH), and under continued TH action, the remaining epithelial cells dedifferentiate into stem cells (SCs), which then newly generate an adult epithelium analogous to the mammalian intestinal epithelium. Previously, we have shown that the precursors of the SCs that exist in the larval epithelium as differentiated absorptive cells specifically express receptor tyrosine kinase-like orphan receptor 2 (Ror2). By using Ror2 as a marker, we have immunohistochemically shown here that these SC precursors, but not the larval epithelial cells destined to die by apoptosis, express TH receptor α (TRα). Upon initiation of TH-dependent remodeling, TRα expression remains restricted to the SCs as well as proliferating adult epithelial primordia derived from them. As intestinal folds form, TRα expression becomes localized in the trough of the folds where the SCs reside. In contrast, TRß expression is transiently up-regulated in the entire intestine concomitantly with the increase of endogenous TH levels and is most highly expressed in the developing adult epithelial primordia. Moreover, we have shown here that global histone H4 acetylation is enhanced in the SC precursors and adult primordia including the SCs, while tri-methylation of histone H3 lysine 27 is lacking in those cells during metamorphosis. Our results strongly suggest distinct roles of TRα and TRß in the intestinal larval-to-adult remodeling, involving distinctive epigenetic modifications in the SC lineage.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Intestinos/crescimento & desenvolvimento , Metamorfose Biológica/genética , Receptores dos Hormônios Tireóideos/metabolismo , Células-Tronco/citologia , Xenopus laevis/genética , Acetilação , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histonas/metabolismo , Larva/metabolismo , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores dos Hormônios Tireóideos/genética , Células-Tronco/metabolismo , Fatores de Tempo , Xenopus laevis/metabolismo
16.
J Hazard Mater ; 381: 120995, 2020 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-31425913

RESUMO

Despite the great concerns associated with the combined biological effects of nanoparticles and insecticides, the current understanding of the corresponding ecological risks remains limited. Xenopus laevis (X. laevis) tadpoles were exposed to various concentrations of typical pyrethroid (cis-bifenthrin; cis-BF), either alone or in combination with graphene oxide (GO), for 21 days. The presence of GO resulted in increased bioconcentration of cis-BF and a higher 1S-enantiomer fraction. Exposure to cis-BF and GO caused further reduction in pre-metamorphic developmental rates and activated dopaminergic, noradrenergic, and serotonergic neurotransmitter systems. Reduced tadpole activity and levels of genomic DNA methylation at cytosine nucleotides (5hmC) were observed in the coexposure groups. These results indicate that GO enhance the bioconcentration of cis-BF and promote the conversion of its 1R-enantiomer to the 1S form, which lead to disruption of neurotransmitter systems as well as interference in metamorphic development.


Assuntos
Grafite/toxicidade , Inseticidas/toxicidade , Piretrinas/toxicidade , Aminoácidos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Metilação de DNA , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Locomoção/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Neurotransmissores/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangue , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
17.
Environ Sci Pollut Res Int ; 27(4): 3697-3705, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30835066

RESUMO

Pesticides are often found at high concentrations in small ponds near agricultural field where amphibians are used to live and reproduce. Even if there are many studies on the impacts of phytopharmaceutical active ingredients in amphibian toxicology, only a few are interested in the earlier steps of their life cycle. While their populations are highly threatened with extinction. The aim of this work is to characterize the effects of glyphosate and its commercial formulation Roundup® GT Max on the Xenopus laevis oocyte maturation which is an essential preparation for the laying and the fertilization. Glyphosate is an extensively used herbicide, not only known for its effectiveness but also for its indirect impacts on non-target organisms. Our results showed that exposures to both forms of glyphosate delayed this hormone-dependent process and were responsible for spontaneous maturation. Severe and particular morphogenesis abnormalities of the meiotic spindle were also observed. The MAPK pathway and the MPF did not seem to be affected by exposures. The xenopus oocyte is particularly affected by the exposures and appears as a relevant model for assessing the effects of environmental contamination.


Assuntos
Glicina/análogos & derivados , Glicina/toxicidade , Herbicidas/toxicidade , Oócitos/efeitos dos fármacos , Xenopus laevis/crescimento & desenvolvimento , Animais , Xenopus laevis/metabolismo
18.
Methods Mol Biol ; 2041: 87-106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646482

RESUMO

Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.


Assuntos
5'-Nucleotidase/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , Receptores Purinérgicos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , 5'-Nucleotidase/genética , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Feminino , Sondas RNA , Receptores Purinérgicos/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
19.
Insect Biochem Mol Biol ; 117: 103289, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31778795

RESUMO

The Xenopus oocyte and the Human Embryonic Kidney (HEK) 293 cell expression systems are frequently used for functional characterization (deorphanization) of insect odorant receptors (ORs). However, the inherent characteristics of these heterologous systems differ in several aspects, which raises the question of whether the two systems provide comparable results, and how well the results correspond to the responses obtained from olfactory sensory neurons in vivo. Five candidate pheromone receptors were previously identified in the primitive moth Eriocrania semipurpurella (Esem) and their responses were characterized in HEK cells. We re-examined the responses of these five EsemORs in Xenopus oocytes. We showed that in both systems, EsemOR1 specifically responded to the plant volatile ß-caryophyllene. EsemOR3 responded stronger to the pheromone component (S,Z)-6-nonen-2-ol than to its enantiomer (R,Z)-6-nonen-2-ol, the second pheromone component. However, EsemOR3 also responded secondarily to the plant volatile ß-caryophyllene in the oocyte system, but not in the HEK cell system. EsemOR4 was unresponsive in the HEK cells, but responded primarily to (R,Z)-6-nonen-2-ol followed by (S,Z)-6-nonen-2-ol in the oocytes, representing a discovery of a new pheromone receptor in this species. EsemOR5 was broadly tuned in both systems, but the rank order among the most active pheromone compounds and antagonists was different. EsemOR6 showed no response to any compound in either system. We compared the results obtained in the two different heterologous systems with the activity previously recorded in vivo, and performed in situ hybridization to localize the expression of these OR genes in the antennae. In spite of similar results overall, differences in OR responses between heterologous expression systems suggest that conclusions about the function of individual ORs may differ depending on the system used for deorphanization.


Assuntos
Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Receptores Odorantes/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Células HEK293 , Humanos , Masculino , Oócitos , Xenopus laevis/metabolismo
20.
Chemosphere ; 243: 125288, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31743868

RESUMO

The decrease in the population of amphibians all over the world has raised concerns. Adult X. laevis frogs were exposed to 0, 1 and 10 mg/L triadimefon and triadimenol. After 14 or 28 days exposure, we collected male and female specimens to study swimming activity, lactic dehydrogenase (LDH) and antioxidant enzyme activity in blood samples, histopathology of liver and thyroid tissue, thyroid hormone levels and thyroid hormone-related gene expression levels in brains. Our results showed that triadimefon and triadimenol could affect the swimming activity of frogs and that this was distinct at different levels of triadimenol. Moreover, triadimefon and triadimenol exposure produced a greater effect on superoxide dismutase (SOD) in females than in males, which was reverse to the finding for glutathione S-transferase (GST) and catalase (CAT). After 28 days exposure, triadimefon produced more toxic effects on the liver than observed for triadimenol. Besides this, triadimefon and triadimenol exposure exerted a greater effect on liver histology and thyroid hormone levels in male frogs than in the females. Our results also found that the expression of genes related to thyroid hormone in brains depended on the exposure level and time, as well as the sex of the treated individual. This study shed light on the relationships between the toxicity of metabolite products and their parent compounds and provided further understanding of the risk of pesticide use on amphibians.


Assuntos
Fungicidas Industriais/toxicidade , Triazóis/toxicidade , Animais , Feminino , Fígado/metabolismo , Masculino , Testes de Toxicidade , Xenopus laevis/metabolismo , Xenopus laevis/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA