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1.
Environ Toxicol Pharmacol ; 87: 103738, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34492396

RESUMO

The effects of two drugs containing the synthetic thyroid hormone levothyroxine (LEV) and an anti-thyroid drug containing propylthiouracil (PTU) on the three early life stages of Xenopus laevis were evaluated with the Frog Embryo Teratogenesis Assay-Xenopus, Tadpole Toxicity Test, and Amphibian Metamorphosis Assay using biochemical and morphological markers. Tested drugs caused more effective growth retardation in stage 8 embryos than stage 46 tadpoles. Significant inhibition of biomarker enzymes has been identified in stage 46 tadpoles for both drugs. AMA test results showed that LEV-I caused progression in the developmental stage and an increase in thyroxine level in 7 days exposure and growth retardation in 21 days exposure in stage 51 tadpoles. On the other hand, increases in lactate dehydrogenase activity for both drugs in the AMA test may be due to impacted energy metabolism during sub-chronic exposure. These results also show that the sensitivity and responses of Xenopus laevis at different early developmental stages may be different when exposed to drugs.


Assuntos
Antitireóideos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Larva/efeitos dos fármacos , Propiltiouracila/toxicidade , Teratógenos/toxicidade , Tiroxina/toxicidade , Xenopus laevis , Acetilcolinesterase/metabolismo , Animais , Carboxilesterase/metabolismo , Embrião não Mamífero/anormalidades , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Larva/enzimologia , Larva/crescimento & desenvolvimento , Masculino , Metamorfose Biológica/efeitos dos fármacos , Xenopus laevis/anormalidades , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
2.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576043

RESUMO

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.


Assuntos
Inseticidas/farmacologia , Neonicotinoides/farmacologia , Antagonistas Nicotínicos/farmacologia , Nitrocompostos/farmacologia , Tiazinas/farmacologia , Animais , Baratas/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Receptores Nicotínicos , Xenopus laevis
3.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502237

RESUMO

Neural crest (NC) cells are highly migratory cells that contribute to various vertebrate tissues, and whose migratory behaviors resemble cancer cell migration and invasion. Information exchange via dynamic NC cell-cell contact is one mechanism by which the directionality of migrating NC cells is controlled. One transmembrane protein that is most likely involved in this process is protein tyrosine kinase 7 (PTK7), an evolutionary conserved Wnt co-receptor that is expressed in cranial NC cells and several tumor cells. In Xenopus, Ptk7 is required for NC migration. In this study, we show that the Ptk7 protein is dynamically localized at cell-cell contact zones of migrating Xenopus NC cells and required for contact inhibition of locomotion (CIL). Using deletion constructs of Ptk7, we determined that the extracellular immunoglobulin domains of Ptk7 are important for its transient accumulation and that they mediate homophilic binding. Conversely, we found that ectopic expression of Ptk7 in non-NC cells was able to prevent NC cell invasion. However, deletion of the extracellular domains of Ptk7 abolished this effect. Thus, Ptk7 is sufficient at protecting non-NC tissue from NC cell invasion, suggesting a common role of PTK7 in contact inhibition, cell invasion, and tissue integrity.


Assuntos
Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Movimento Celular , Inibição de Contato , Neoplasias Pulmonares/metabolismo , Crista Neural/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Polaridade Celular , Humanos , Neoplasias Pulmonares/patologia , Xenopus laevis
4.
Nat Commun ; 12(1): 5482, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531379

RESUMO

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.


Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Xenopus laevis/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas/genética , Animais , Desenvolvimento Embrionário/genética , Camundongos , Estabilidade de RNA/genética , Xenopus laevis/embriologia , Peixe-Zebra/embriologia
5.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548398

RESUMO

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci (ift80 and ift172) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies.


Assuntos
Osso e Ossos/anormalidades , Ciliopatias/patologia , Craniossinostoses/patologia , Proteínas do Citoesqueleto/metabolismo , Displasia Ectodérmica/patologia , Embrião não Mamífero/patologia , Anormalidades Musculoesqueléticas/patologia , Doenças Renais Policísticas/patologia , Tubulina (Proteína)/química , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Ciliopatias/genética , Ciliopatias/metabolismo , Craniossinostoses/genética , Craniossinostoses/metabolismo , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Embrião não Mamífero/metabolismo , Anormalidades Musculoesqueléticas/genética , Anormalidades Musculoesqueléticas/metabolismo , Fenótipo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Tubulina (Proteína)/metabolismo , Xenopus laevis
6.
Nat Commun ; 12(1): 5282, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489418

RESUMO

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell's endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Homeostase/genética , Modelos Estatísticos , Neuroglia/metabolismo , Células A549 , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/genética , Animais , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Simulação por Computador , Expressão Gênica , Humanos , Cinética , Metabolômica/métodos , Neuroglia/citologia , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis
7.
Nat Commun ; 12(1): 5280, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489435

RESUMO

Little is known about the roles of histone tails in modulating nucleosomal DNA accessibility and its recognition by other macromolecules. Here we generate extensive atomic level conformational ensembles of histone tails in the context of the full nucleosome, totaling 65 microseconds of molecular dynamics simulations. We observe rapid conformational transitions between tail bound and unbound states, and characterize kinetic and thermodynamic properties of histone tail-DNA interactions. Different histone types exhibit distinct binding modes to specific DNA regions. Using a comprehensive set of experimental nucleosome complexes, we find that the majority of them target mutually exclusive regions with histone tails on nucleosomal/linker DNA around the super-helical locations ± 1, ± 2, and ± 7, and histone tails H3 and H4 contribute most to this process. These findings are explained within competitive binding and tail displacement models. Finally, we demonstrate the crosstalk between different histone tail post-translational modifications and mutations; those which change charge, suppress tail-DNA interactions and enhance histone tail dynamics and DNA accessibility.


Assuntos
DNA/química , Histonas/química , Nucleossomos/ultraestrutura , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/química , Animais , Sítios de Ligação , DNA/genética , DNA/metabolismo , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Eletricidade Estática , Transcrição Genética , Xenopus laevis
8.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544871

RESUMO

Molecular and structural facets of cell-cell adhesion have been extensively studied in monolayered epithelia. Here, we perform a comprehensive analysis of cell-cell contacts in a series of multilayered tissues in the Xenopus gastrula model. We show that intercellular contact distances range from 10 to 1,000 nm. The contact width frequencies define tissue-specific contact spectra, and knockdown of adhesion factors modifies these spectra. This allows us to reconstruct the emergence of contact types from complex interactions of the factors. We find that the membrane proteoglycan Syndecan-4 plays a dominant role in all contacts, including narrow C-cadherin-mediated junctions. Glypican-4, hyaluronic acid, paraxial protocadherin, and fibronectin also control contact widths, and unexpectedly, C-cadherin functions in wide contacts. Using lanthanum staining, we identified three morphologically distinct forms of glycocalyx in contacts of the Xenopus gastrula, which are linked to the adhesion factors examined and mediate cell-cell attachment. Our study delineates a systematic approach to examine the varied contributions of adhesion factors individually or in combinations to nondiscrete and seemingly amorphous intercellular contacts.


Assuntos
Caderinas/metabolismo , Adesão Celular , Comunicação Celular , Embrião não Mamífero/fisiologia , Gástrula/fisiologia , Proteínas de Xenopus/metabolismo , Animais , Caderinas/genética , Embrião não Mamífero/citologia , Gástrula/citologia , Glicocálix/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis
9.
EMBO Rep ; 22(9): e50932, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34427977

RESUMO

Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue-specific response to injury remains poorly understood. Using a combination of bulk and single-cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neural progenitors but regulates their fate after division. In foxm1-/- tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord-specific response to injury and reveal a new role for neuronal differentiation during regeneration.


Assuntos
Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Animais , Regulação da Expressão Gênica , Larva , Medula Espinal , Traumatismos da Medula Espinal/genética , Xenopus laevis/genética
10.
Elife ; 102021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34406118

RESUMO

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.


Assuntos
Cromatina/metabolismo , Cromossomos/genética , DNA Topoisomerases Tipo II/genética , Histonas/genética , Adenosina Trifosfatases/metabolismo , Animais , Extratos Celulares/química , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Feminino , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Oócitos/química , Oócitos/metabolismo , Fuso Acromático/genética , Fuso Acromático/patologia , Fuso Acromático/ultraestrutura , Xenopus laevis
11.
J Vis Exp ; (173)2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34369935

RESUMO

The two arms of the Transforming Growth Factor ß (Tgfß) superfamily, represented by Tgfß/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgfß family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgfß receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgfß precursor proteins. RNA encoding Tgfß precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions.


Assuntos
Proteínas Morfogenéticas Ósseas , Fator de Crescimento Transformador beta , Animais , Blastocisto/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Transformador beta/genética , Fatores de Crescimento Transformadores , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
12.
Bull Tokyo Dent Coll ; 62(3): 171-180, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34393144

RESUMO

Bone marrow is the principal site of hematopoiesis in mammals. Amphibians were the first phylogenetic group in vertebrates to acquire bone marrow, but the distribution of hematopoietic cells in the bone marrow of the primitive frog, Xenopus laevis (X. laevis) has not been well documented. The purpose of this study was to perform a histological investigation of the distribution of hematopoietic cells in femoral bone marrow at various stages of development in X. laevis. Hematopoietic cells showed preferential distribution on the endosteal surface of cortical bone throughout all stages of development, from tadpole to aged frog. In mature frogs, hematopoietic cells appeared at the boundary between the epiphysis and the bone marrow. The distribution of hematopoietic cells around the blood vessels was limited to a small number of vessels in the bone marrow. Abundant adipose tissue was observed in the bone marrow cavity from the tadpole stage to the mature frog stage. Hematopoietic cells showed preferential distribution in a belt-like fashion on the surface of newly-formed bones in a bone regeneration model in the diaphysis of X. laevis. These results indicate that the distribution of hematopoietic cells in bone marrow in X. laevis differs from that in mammals, and that the bone marrow of X. laevis constitutes a useful model for exploring the mechanism underlying the phylogenetic differentiation of bone marrow hematopoiesis.


Assuntos
Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Animais , Hematopoese , Filogenia , Xenopus laevis
13.
Neuron ; 109(18): 2902-2913.e4, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34390650

RESUMO

TRAAK is a mechanosensitive two-pore domain K+ (K2P) channel localized to nodes of Ranvier in myelinated neurons. TRAAK deletion in mice results in mechanical and thermal allodynia, and gain-of-function mutations cause the human neurodevelopmental disorder FHEIG. TRAAK displays basal and stimulus-gated activities typical of K2Ps, but the mechanistic and structural differences between these modes are unknown. Here, we demonstrate that basal and mechanically gated openings are distinguished by their conductance, kinetics, and structure. Basal openings are low conductance, short duration, and due to a conductive channel conformation with the interior cavity exposed to the surrounding membrane. Mechanically gated openings are high conductance, long duration, and due to a channel conformation in which the interior cavity is sealed to the surrounding membrane. Our results explain how dual modes of activity are produced by a single ion channel and provide a basis for the development of state-selective pharmacology with the potential to treat disease.


Assuntos
Ativação do Canal Iônico/fisiologia , Mecanotransdução Celular/fisiologia , Neurônios/fisiologia , Canais de Potássio/química , Canais de Potássio/fisiologia , Animais , Feminino , Humanos , Estimulação Física/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomycetales , Xenopus laevis
14.
J Exp Biol ; 224(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34424964

RESUMO

Characterizing sex and species differences in muscle physiology can contribute to a better understanding of proximate mechanisms underlying behavioral evolution. In Xenopus, the laryngeal muscle's ability to contract rapidly and its electromyogram potentiation allows males to produce calls that are more rapid and intensity-modulated than female calls. Prior comparative studies have shown that some species lacking typical male features of vocalizations sometimes show reduced sex differences in underlying laryngeal physiology. To further understand the evolution of sexually differentiated laryngeal muscle physiology and its role in generating behavior, we investigated sex differences in the laryngeal physiology of X. muelleri, a species in which male and female calls are similar in rapidity but different with respect to intensity modulation. We delivered ethologically relevant stimulus patterns to ex vivo X. muelleri larynges to investigate their ability to produce various call patterns, and we also delivered stimuli over a broader range of intervals to assess sex differences in muscle tension and electromyogram potentiation. We found a small but statistically significant sex difference in laryngeal electromyogram potentiation that varied depending on the number of stimuli. We also found a small interaction between sex and stimulus interval on muscle tension over an ethologically relevant range of stimulus intervals; male larynges were able to produce similar tensions to female larynges at slightly smaller (11-12 ms) inter-stimulus intervals. These findings are consistent with behavioral observations and present a previously undescribed intermediate sex difference in Xenopus laryngeal muscle physiology.


Assuntos
Laringe , Caracteres Sexuais , Animais , Feminino , Masculino , Vocalização Animal , Xenopus , Xenopus laevis
15.
J Exp Biol ; 224(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34424980

RESUMO

Environmental temperature variation generates adaptive phenotypic differentiation in widespread populations. We used a common garden experiment to determine whether offspring with varying parental origins display adaptive phenotypic variation related to different thermal conditions experienced in parental environments. We compared burst swimming performance and critical thermal limits of African clawed frog (Xenopus laevis) tadpoles bred from adults captured at high (∼2000 m above sea level) and low (∼ 5 m above sea level) altitudes. Maternal origin significantly affected swimming performance. Optimal swimming performance temperature (Topt) had a >9°C difference between tadpoles with low altitude maternal origins (pure- and cross-bred, 35.0°C) and high-altitude maternal origins (pure-bred, 25.5°C; cross-bred, 25.9°C). Parental origin significantly affected critical thermal (CT) limits. Pure-bred tadpoles with low-altitude parental origins had higher CTmax (37.8±0.8°C) than pure-bred tadpoles with high-altitude parental origins and all cross-bred tadpoles (37.0±0.8 and 37.1±0.8°C). Pure-bred tadpoles with low-altitude parental origins and all cross-bred tadpoles had higher CTmin (4.2±0.7 and 4.2±0.7°C) than pure-bred tadpoles with high-altitude parental origins (2.5±0.6°C). Our study shows that the varying thermal physiological traits of Xenopus laevis tadpoles are the result of adaptive responses to their parental thermal environments. This study is one of few demonstrating potential intraspecific evolution of critical thermal limits in a vertebrate species. Multi-generation common garden experiments and genetic analyses would be required to further tease apart the relative contribution of plastic and genetic effects to the adaptive phenotypic variation observed in these tadpoles.


Assuntos
Altitude , Natação , Animais , Larva , Temperatura , Xenopus laevis/genética
16.
Nat Commun ; 12(1): 4709, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354080

RESUMO

Allostery represents a fundamental mechanism of biological regulation that involves long-range communication between distant protein sites. It also provides a powerful framework for novel therapeutics. NMDA receptors (NMDARs), glutamate-gated ionotropic receptors that play central roles in synapse maturation and plasticity, are prototypical allosteric machines harboring large extracellular N-terminal domains (NTDs) that provide allosteric control of key receptor properties with impact on cognition and behavior. It is commonly thought that GluN2A and GluN2B receptors, the two predominant NMDAR subtypes in the adult brain, share similar allosteric transitions. Here, combining functional and structural interrogation, we reveal that GluN2A and GluN2B receptors utilize different long-distance allosteric mechanisms involving distinct subunit-subunit interfaces and molecular rearrangements. NMDARs have thus evolved multiple levels of subunit-specific allosteric control over their transmembrane ion channel pore. Our results uncover an unsuspected diversity in NMDAR molecular mechanisms with important implications for receptor physiology and precision drug development.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fotoquímica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
17.
Nat Commun ; 12(1): 4680, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344887

RESUMO

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10-10), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases.


Assuntos
Síndrome de Goldenhar/genética , Haploinsuficiência , Fatores de Processamento de RNA/genética , Adolescente , Adulto , Animais , Criança , Exoma/genética , Feminino , Estudos de Associação Genética , Síndrome de Goldenhar/patologia , Humanos , Lactente , Masculino , Mutação , Crista Neural/crescimento & desenvolvimento , Crista Neural/patologia , Linhagem , Spliceossomos/genética , Xenopus laevis
18.
Elife ; 102021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34213414

RESUMO

In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in Xenopus oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.


Assuntos
Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Regulação da Expressão Gênica
19.
Phytomedicine ; 90: 153646, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34280827

RESUMO

BACKGROUND: Gamma-aminobutyric acid A (GABAA) receptors have been implicated in anxiety and epileptic disorders. HYPOTHESIS/PURPOSE: This study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders. METHODS: Stigmasterol was evaluated on various recombinant GABAA receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2ß2γ2L, α4ß3δ, and α4ß3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABAA α2ß2γ2L, α4ß3, and α4ß3δ complexes by in silico docking. RESULTS: Stigmasterol enhanced GABA-induced currents at ternary α2ß2γ2L, α4ß3δ, and binary α4ß3 GABAAR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4ß3δ was significantly higher compared to the binary α4ß3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4ß3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of ß3 at α4ß3δ complex. In in vivo studies, Stigmasterol (0.5-3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism. CONCLUSION: To our knowledge, this is the first study reporting the positive modulation of GABAA receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.


Assuntos
Ansiolíticos , Anticonvulsivantes/farmacologia , Moduladores GABAérgicos/farmacologia , Estigmasterol , Animais , Ansiolíticos/farmacologia , Oócitos , Receptores de GABA-A , Convulsões/tratamento farmacológico , Estigmasterol/farmacologia , Xenopus laevis
20.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199759

RESUMO

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.


Assuntos
Alelos , Deficiência Intelectual/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Canais de Potássio/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcineurina/metabolismo , Feminino , Genoma Humano , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ionomicina/farmacologia , Masculino , Linhagem , Canais de Potássio/química , Irmãos , Xenopus laevis/metabolismo , Adulto Jovem
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