Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24.702
Filtrar
1.
Rev Environ Contam Toxicol ; 252: 131-171, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463624

RESUMO

Anurans from the genus Xenopus have long been used as standard testing organisms and occur naturally in tropical and sub-tropical areas where malaria vector control pesticides are actively used. However, literature on the toxic effects of these pesticides is limited. This review analyses the available data pertaining to both Xenopus and the pesticides used for malaria vector control in order to determine the pesticides that have the greatest potential to influence amphibian health while also identifying gaps in literature that need to be addressed. Amphibian diversity has shown the fastest decline of any group, yet there are still voids in our understanding of how this is happening. The lack of basic toxicity data on amphibians with regard to pesticides is an issue that needs to be addressed in order to improve effectiveness of amphibian conservation strategies. Meta-analyses performed in this review show that, at current usage, with the available acute toxicity literature, the pyrethroid pesticide group could hold the highest potential to cause acute toxicity to Xenopus sp. in relation to the other MVCPs discussed, but the lack of data cripples the efficacy with which meta-analyses can be performed and conclusions made from such analyses. Several studies have shown that DDT accumulates in Xenopus sp. from malaria vector control areas, but accumulation of other MVCPs in frogs is still largely unknown. Through this review we hope to encourage future research into the field of amphibian ecotoxicology and to promote the use of the Xenopus standard model in order to build comprehensive datasets that may be used in amphibian conservation.


Assuntos
Ecotoxicologia , Poluentes Ambientais/toxicidade , Malária , Controle de Mosquitos , Mosquitos Vetores , Praguicidas/toxicidade , Animais , Anopheles , Xenopus , Xenopus laevis
2.
Science ; 366(6465): 569-570, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672880
3.
Results Probl Cell Differ ; 68: 379-418, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31598865

RESUMO

The developmental adaptations of the marsupial frogs Gastrotheca riobambae and Flectonotus pygmaeus (Hemiphractidae) are described and compared with frogs belonging to seven additional families. Incubation of embryos by the mother in marsupial frogs is associated with changes in the anatomy and physiology of the female, modifications of oogenesis, and extraordinary changes in embryonic development. The comparison of early development reveals that gene expression is highly conserved. However, the timing of gene expression varies between frog species. There are two modes of gastrulation according to the onset of convergent extension. In gastrulation mode 1, convergent extension is an intrinsic mechanism of gastrulation. This gastrulation mode occurs in frogs with aquatic reproduction, such as Xenopus laevis. In gastrulation mode 2, convergent extension occurs after the completion of gastrulation movements. Gastrulation mode 2 occurs in frogs with terrestrial reproduction, such as the marsupial frog, G. riobambae. The two modes of frog gastrulation resemble the two transitions toward meroblastic cleavage of ray-finned fishes (Actinopterygii). The comparison indicates that a major event in the evolution of frog terrestrial development is the separation of convergent extension from gastrulation.


Assuntos
Anuros/embriologia , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Animais , Gástrula/embriologia , Xenopus laevis/embriologia
4.
Chem Commun (Camb) ; 55(75): 11215-11218, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31469130

RESUMO

Here we report the dephosphorylation and proteolysis of phosphorylated α-synuclein, a Parkinson's disease-related protein, in living cells in a time resolved manner using in-cell NMR.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Oócitos/metabolismo , alfa-Sinucleína/metabolismo , Animais , Oócitos/química , Fosforilação , Proteólise , Xenopus laevis , alfa-Sinucleína/química
5.
Arch Environ Contam Toxicol ; 77(3): 390-408, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31422435

RESUMO

One of the direct causes of biodiversity loss is environmental pollution resulting from the use of chemicals. Different kinds of chemicals, such as persistent organic pollutants and some heavy metals, can be endocrine disruptors, which act at low doses over a long period of time and have a negative effect on the reproductive and thyroid system in vertebrates worldwide. Research on the effects of endocrine disruptors and the use of bioindicators in neotropical ecosystems where pressure on biodiversity is high is scarce. In Chile, although endocrine disruptors have been detected at different concentrations in the environments of some ecosystems, few studies have been performed on their biological effects in the field. In this work, Xenopus laevis (African clawed frog), an introduced species, is used as a bioindicator for the presence of endocrine disruptors in aquatic systems with different degrees of contamination in a Mediterranean zone in central Chile. For the first time for Chile, alterations are described that can be linked to exposure to endocrine disruptors, such as vitellogenin induction, decreased testosterone in male frogs, and histological changes in gonads. Dioxin-like and oestrogenic activity was detected in sediments at locations where it seem to be related to alterations found in the frogs. In addition, an analysis of land use/cover use revealed that urban soil was the best model to explain the variations in frog health indicators. This study points to the usefulness of an invasive species as a bioindicator for the presence of endocrine-disruptive chemicals.


Assuntos
Disruptores Endócrinos/toxicidade , Biomarcadores Ambientais , Exposição Ambiental/análise , Poluição Ambiental/efeitos adversos , Xenopus laevis/fisiologia , Animais , Linhagem Celular Tumoral , Chile , Ecossistema , Ecotoxicologia/métodos , Disruptores Endócrinos/análise , Poluentes Ambientais/toxicidade , Feminino , Sedimentos Geológicos/análise , Gônadas/patologia , Humanos , Espécies Introduzidas , Masculino , Reprodução , Testosterona/metabolismo , Vitelogeninas/metabolismo
6.
Chemosphere ; 235: 952-958, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31299708

RESUMO

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (rs) = 0.88], body weight (rs = 0.86), and length (rs = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages.


Assuntos
Cyprinidae/embriologia , Metamorfose Biológica/efeitos dos fármacos , Oncorhynchus mykiss/embriologia , Praguicidas/toxicidade , Poluentes Químicos da Água/toxicidade , Xenopus laevis/embriologia , Animais , Ecologia , Larva/efeitos dos fármacos , Reprodução , Medição de Risco/métodos , Alimentos Marinhos
7.
Aquat Toxicol ; 214: 105237, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31276910

RESUMO

Chirality should be taken into consideration when assessing the effect of synthetic pyrethroids to aquatic environmental safety. In our study, 96 h acute toxicity assay showed that enantiomers of cis-BF had an addictive effect of toxicity on Xenopus laevis and R-cis-bifenthrin(R-cis-BF) had higher acute toxicity than S-cis-BF. In chronic assay, R-cis-BF exerted more toxic effect on behavior and development of tadpoles than S-cis-BF, and there was also enantioselective effect of cis-BF on antioxidant enzyme and LDH activity. Besides, thyroid development was also affected at the gene and hormone level, with varied effects observed with different exposure enantiomers. Moreover, in the enantioselective accumulation and tissue distribution of enantiomer assays, results showed that R-cis-BF had higher affinity to organisms than S-cis-BF. This study provided the evidence that chiral pesticides enantioselectively affected development of amphibians, and also shed light on the understanding of enantioselectivity in both acute and chronic eco-toxicities to improve risk assessment and regulation of chiral pesticides.


Assuntos
Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Piretrinas/química , Piretrinas/toxicidade , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo , Animais , Antioxidantes/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inseticidas/toxicidade , L-Lactato Desidrogenase/metabolismo , Larva/efeitos dos fármacos , Malondialdeído/metabolismo , Piretrinas/metabolismo , Estereoisomerismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidade
8.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
9.
Aquat Toxicol ; 213: 105218, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203168

RESUMO

The toxic effects of different atrazine concentrations on tadpoles and adult male African clawed frogs (Xenopus laevis) were assessed in a controlled laboratory environment following 90 days' exposure. The aim was to elucidate the danger of atrazine exposure on the cardiac tissue relative to its critical function of rhythmic contractility, fundamental for optimal blood circulation and homeostasis. Tadpoles and adult frogs were exposed to 0 µg/L (control), 0.01 µg L-1, 200 µg L-1 and 500 µg L-1 concentrations of atrazine for 90 days. Mortality was concenration-dependent and significantly increased in juvenile group (77%, 43%, 23% and 0 respectively for 500 µg L-1, 200 µg L-1, 0.01 µg L-1, and control group). While the mean juvenile heart area decreased concentration-dependently, adult frog mean heart area significantly increased in the 200 µg L-1 group only and mean heart weight change was variable across all exposure levels. Light microscopy of hematoxylin and eosin (H&E) and Mallory-Heidenhain rapid one-step staining techniques on cardiac tissue sections of the juvenile and adult frogs revealed shrinkage of cardiac muscle cells into thin wavy myocytes. Additionally, disorganized branching of muscle fibres with reduced striations were observed in 0.01 µg L-1 and 200 µg L-1 but hypertrophied myocytes, thickened intensely staining myofibrils in the 500 µg L-1 group in juvenile and adult frogs. Significant increase in the mean percentage area of connective tissue in all the treated groups (p < 0.036) were also recorded. Immunohistochemistry analysis showed decreased eNOS localization in cardiac tissue in 200 µg L-1 and 500 µg L-1 of both juvenile and adult group, suggestive of decreased cardiac contractility due to atrazine exposure. The results indicate that environmentally relevant atrazine concentrations cause significant mortality in tadpoles while concentrations ≥200 µg L-1 adversely affect cardiac muscle morphology and may induce functional perturbations in cardiac tissue contractility and consequent dysfunction which generally may have an adverse impact on their survival and longevity.


Assuntos
Atrazina/toxicidade , Cardiotoxicidade/patologia , Poluentes Químicos da Água/toxicidade , Xenopus laevis/fisiologia , Animais , Tecido Conjuntivo/efeitos dos fármacos , Coração/efeitos dos fármacos , Larva/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Análise de Sobrevida
10.
Nucleic Acids Res ; 47(10): 5436-5448, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31162607

RESUMO

HDGF-related protein 3 (HRP3, also known as HDGFL3) belongs to the family of HDGF-related proteins (HRPs) and plays an essential role in hepatocellular carcinoma pathogenesis. All HRPs have a PWWP domain at the N-terminus that binds both histone and DNA substrates. Despite previous advances in PWWP domains, the molecular basis by which HRP3 interacts with chromatin is unclear. In this study, we solved the crystal structures of the HRP3 PWWP domain in complex with various double-stranded DNAs with/without bound histone peptides. We found that HRP3 PWWP bound to the phosphate backbone of the DNA minor groove and showed a preference for DNA molecules bearing a narrow minor groove width. In addition, HRP3 PWWP preferentially bound to histone peptides bearing the H3K36me3/2 modification. HRP3 PWWP uses two adjacent surfaces to bind both DNA and histone substrates simultaneously, enabling us to generate a model illustrating the recruitment of PWWP to H3K36me3-containing nucleosomes. Cell-based analysis indicated that both DNA and histone binding by the HRP3 PWWP domain is important for HRP3 recruitment to chromatin in vivo. Our work establishes that HRP3 PWWP is a new family of minor groove-specific DNA-binding proteins, which improves our understanding of HRP3 and other PWWP domain-containing proteins.


Assuntos
Cromatina/química , DNA/química , Proteínas Nucleares/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Células Hep G2 , Histonas/química , Humanos , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Nucleossomos/química , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Frações Subcelulares , Xenopus laevis
11.
Mar Drugs ; 17(6)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181805

RESUMO

In order to improve stability of a peptide marine drug lead, α-conotoxin TxID, we synthesized and modified TxID at the N-terminal with DSPE-PEG-NHS by a nucleophilic substitution reaction to prepare the DSPE-PEG-TxID for the first time. The reaction conditions, including solvent, ratio, pH, and reaction time, were optimized systematically and the optimal one was reacted in dimethyl formamide at pH 8.2 with triethylamine at room temperature for 120 h. The in vitro stabilities in serum, simulated gastric juice, and intestinal fluid were tested, and improved dramatically compared with TxID. The PEG-modified peptide was functionally tested on α3ß4 nicotinic acetylcholine receptor (nAChR) heterologously expressed in Xenopus laevis oocytes. The DSPE-PEG-TxID showed an obvious inhibition effect on α3ß4 nAChR. All in all, the PEG modification of TxID was improved in stability, resistance to enzymatic degradation, and may prolong the half-life in vivo, which may pave the way for the future application in smoking cessation and drug rehabilitation, as well as small cell lung cancer.


Assuntos
Conotoxinas/metabolismo , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Animais , Conotoxinas/química , Estabilidade de Medicamentos , Embrião não Mamífero , Humanos , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Receptores Nicotínicos/metabolismo , Xenopus laevis
12.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035589

RESUMO

BACKGROUND: Astaxanthin (ATX) is a lipophilic compound found in many marine organisms. Studies have shown that ATX has many strong biological properties, including antioxidant, antiviral, anticancer, cardiovascular, anti-inflammatory, neuro-protective and anti-diabetic activities. However, no research has elucidated the effect of ATX on ionic channels. ATX can be extracted from shrimp by-products. Our work aims to characterize ATX cell targets to lend value to marine by-products. METHODS: We used the Xenopus oocytes cell model to characterize the pharmacological target of ATX among endogenous Xenopus oocytes' ionic channels and to analyze the effects of all carotenoid-extract samples prepared from shrimp by-products using a supercritical fluid extraction (SFE) method. RESULTS: ATX inhibits amiloride-sensitive sodium conductance, xINa, in a dose-dependent manner with an IC50 of 0.14 µg, a maximum inhibition of 75% and a Hill coefficient of 0.68. It does not affect the potential of half activation, but significantly changes the kinetics, according to the slope factor values. The marine extract prepared from shrimp waste at 10 µg inhibits xINa in the same way as ATX 0.1 µg does. When ATX was added to the entire extract at 10 µg, inhibition reached that induced with ATX 1 µg. CONCLUSIONS: ATX and the shrimp Extract inhibit amiloride-sensitive sodium channels in Xenopus oocytes and the TEVC method makes it possible to measure the ATX inhibitory effect in bioactive SFE-Extract samples.


Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Oócitos/fisiologia , Amilorida/farmacologia , Animais , Descoberta de Drogas/métodos , Canais de Sódio/metabolismo , Xenopus laevis
13.
Methods Mol Biol ; 1966: 225-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041751

RESUMO

This chapter describes a technique that can be used to isolate adipose tissue macrophages (ATMs) from the visceral white adipose tissue. Nevertheless, this technique can also be used to isolate ATMs from subcutaneous white adipose tissue and brown adipose tissue from mouse, human subcutaneous fat depot, and also from the fat body of the toad Xenopus. We detail the flow-cytometric gating strategy that has been developed to identify ATM population, and we describe the isolation of RNA from this population and its use for gene expression profiling. Finally, we describe in vitro culture of ATMs for downstream applications.


Assuntos
Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Macrófagos , Tecido Adiposo Marrom/citologia , Animais , Humanos , Camundongos , Gordura Subcutânea/citologia , Xenopus laevis
14.
BMC Genomics ; 20(1): 386, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101013

RESUMO

BACKGROUND: Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development. RESULTS: We find that SUMOylation-deficient embryos consistently exhibit defects in neural tube and heart development. We have measured differences in gene expression between control and embryos injected with Gam1 mRNA at three developmental stages: early gastrula (immediately following the initiation of zygotic transcription), late gastrula (completion of the formation of the three primary germ layers), and early neurula (appearance of the neural plate). Although changes in gene expression are widespread and can be linked to many biological processes, three pathways, non-canonical Wnt/PCP, snail/twist, and Ets-1, are especially sensitive to the loss of SUMOylation activity and can largely account for the predominant phenotypes of Gam1 embryos. SUMOylation appears to generate different pools of a given transcription factor having different specificities with this post-translational modification involved in the regulation of more complex, as opposed to housekeeping, processes. CONCLUSIONS: We have identified changes in gene expression that underlie the neural tube and heart phenotypes resulting from depressed SUMOylation activity. Notably, these developmental defects correspond to the two most frequently occurring congenital birth defects in humans, strongly suggesting that perturbation of SUMOylation, either globally or of a specific protein, may frequently be the origin of these pathologies.


Assuntos
Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Defeitos do Tubo Neural/genética , Sumoilação , Proteínas de Xenopus/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Feminino , Perfilação da Expressão Gênica , Cardiopatias Congênitas/patologia , Masculino , Defeitos do Tubo Neural/patologia , Proteínas Virais/administração & dosagem , Xenopus laevis
16.
Planta Med ; 85(11-12): 925-933, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127604

RESUMO

A fluorometric imaging plate reader (FLIPR) assay utilizing Chinese hamster ovary (CHO) cells stably transfected with GABAA receptors of α 1 ß 2 γ 2 subunit composition was evaluated and validated for rapid screening of plant extract libraries and efficient localization of active compounds in extracts. Validation was performed with pure compounds and extracts known to contain allosteric GABAA receptor modulators. Plants extracts that had been previously reported as active in an assay using Xenopus laevis oocytes transiently expressing GABAA receptors of α 1 ß 2 γ 2 subunit composition were also active in the FLIPR assay. A protocol for HPLC-based activity profiling was developed, whereby separations of 0.4 - 1.2 mg of extracts on an analytical HPLC column were found to be sufficient for the sensitivity of the bioassay. The protocol successfully localized the activity of known GABAergic natural products, such as magnolol in Magnolia officinalis, valerenic acid in Valeriana officinalis, and piperine in Piper nigrum extract. EC50 values of compounds (magnolol: 4.81 ± 1.0 µM, valerenic acid: 12.56 ± 1.2 µM, and piperine: 5.76 ± 0.7 µM) were found to be comparable or lower than those reported using Xenopus oocyte assays.


Assuntos
Fluorometria/métodos , Extratos Vegetais/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Alcaloides/farmacologia , Animais , Benzodioxóis/farmacologia , Bioensaio/métodos , Compostos de Bifenilo/farmacologia , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetulus , Indenos/farmacologia , Lignanas/farmacologia , Magnolia/química , Oócitos/metabolismo , Piper nigrum/química , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Sesquiterpenos/farmacologia , Valeriana/química , Xenopus laevis
17.
Pharm Res ; 36(6): 84, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30997560

RESUMO

PURPOSE: ß-Hydroxy-ß-methylbutyrate (HMB), a nutritional supplement, elicits anabolic activity in muscle. Here we investigated the mechanism of HMB uptake in muscle cells. METHODS: Murine muscle cells (C2C12) and human mammary epithelial cells (MCF7) were used for uptake. As HMB is a monocarboxylate, focus was on monocarboxylate transporters, monitoring interaction of HMB with H+-coupled lactate uptake, and influence of H+ directly on HMB uptake. Involvement of MCT1-4 was studied using selective inhibitors and gene silencing. Involvement of human Na+/monocarboxylate transporter SMCT1 was also assessed using Xenopus oocytes. RESULTS: H+-coupled lactate uptake was inhibited by HMB in both mammalian cells. HMB uptake was H+-coupled and inhibited by lactate. C2C12 cells expressed MCT1 and MCT4; MCF7 cells expressed MCT1-4; undifferentiated C2C12 cells expressed SMCT1. SMCT1 mediated Na+-coupled HMB transport. Inhibitors of MCT1/4, siRNA-mediated gene silencing, and expression pattern showed that MCT1-4 were responsible only for a small portion of HMB uptake in these cells. CONCLUSION: HMB uptake in C2C12 and MCF7 cells is primarily H+-coupled and inhibited by lactate, but MCT1-4 are only partly responsible for HMB uptake. SMCT1 also transports HMB, but in a Na+-coupled manner. Other, yet unidentified, transporters mediate the major portion of HMB uptake in C2C12 and MCF7 cells.


Assuntos
Suplementos Nutricionais , Transportadores de Ácidos Monocarboxílicos/metabolismo , Valeratos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Células Epiteliais/metabolismo , Inativação Gênica , Humanos , Ácido Láctico/metabolismo , Células MCF-7 , Camundongos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Células Musculares/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Sódio/metabolismo , Xenopus laevis
18.
Sci Total Environ ; 671: 644-654, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-30939317

RESUMO

Although aquatic vertebrates and humans are increasingly exposed to water pollutants associated with unconventional oil and gas extraction (UOG), the long-term effects of these pollutants on immunity remains unclear. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable and sensitive model for evaluating the effects of waterborne pollutants. X. laevis tadpoles were exposed to a mixture of equimass amount of UOG chemicals with endocrine disrupting activity (0.1 and 1.0 µg/L) for 3 weeks, and then long-term effects on immune function at steady state and following viral (FV3) infection was assessed after metamorphosis. Notably, developmental exposure to the mixture of UOG chemicals at the tadpole stage affected metamorphic development and fitness by significantly decreasing body mass after metamorphosis completion. Furthermore, developmental exposure to UOGs resulted in perturbation of immune homeostasis in adult frogs, as indicated by significantly decreased number of splenic innate leukocytes, B and T lymphocytes; and a weakened antiviral immune response leading to increased viral load during infection by the ranavirus FV3. These findings suggest that mixture of UOG-associated waterborne endocrine disruptors at low but environmentally-relevant levels have the potential to induce long-lasting alterations of immune function and antiviral immunity in aquatic vertebrates and ultimately human populations.


Assuntos
Indústrias Extrativas e de Processamento/métodos , Poluentes Químicos da Água/toxicidade , Xenopus laevis/fisiologia , Animais , Disruptores Endócrinos/toxicidade , Monitoramento Ambiental , Homeostase , Imunidade Inata/efeitos dos fármacos , Larva/imunologia , Leucócitos , Metamorfose Biológica , Ranavirus , Xenopus laevis/imunologia , Xenopus laevis/virologia
19.
Nat Commun ; 10(1): 1804, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000703

RESUMO

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Proteínas Desgrenhadas/metabolismo , Domínios PDZ/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Técnicas Biossensoriais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Fosforilação/fisiologia , Análise de Célula Única/métodos , Xenopus laevis
20.
Nat Commun ; 10(1): 1518, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944331

RESUMO

When migrating in vivo, cells are exposed to numerous conflicting signals: chemokines, repellents, extracellular matrix, growth factors. The roles of several of these molecules have been studied individually in vitro or in vivo, but we have yet to understand how cells integrate them. To start addressing this question, we used the cephalic neural crest as a model system and looked at the roles of its best examples of positive and negative signals: stromal-cell derived factor 1 (Sdf1/Cxcl12) and class3-Semaphorins. Here we show that Sdf1 and Sema3A antagonistically control cell-matrix adhesion via opposite effects on Rac1 activity at the single cell level. Directional migration at the population level emerges as a result of global Semaphorin-dependent confinement and broad activation of adhesion by Sdf1 in the context of a biased Fibronectin distribution. These results indicate that uneven in vivo topology renders the need for precise distribution of secreted signals mostly dispensable.


Assuntos
Movimento Celular/fisiologia , Junções Célula-Matriz/fisiologia , Crista Neural/citologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular , Forma Celular/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Junções Célula-Matriz/efeitos dos fármacos , Junções Célula-Matriz/metabolismo , Quimiocina CXCL12/metabolismo , Feminino , Fibronectinas/metabolismo , Masculino , Manganês/metabolismo , Camundongos , Proteínas do Tecido Nervoso/fisiologia , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Receptores CXCR4/metabolismo , Semaforinas/metabolismo , Xenopus laevis/embriologia , Proteínas rac1 de Ligação ao GTP/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA