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Wnt signaling plays an essential role in cellular processes like development, maturation, and function maintenance. Xenopus laevis oocytes are a suitable model to study not only the development but also the function of different receptors expressed in their membranes, like those receptors expressed in the central nervous system (CNS) including Frizzled 7. Here, using frog oocytes and recordings of endogenous membrane currents in a two-electrode path configuration along with morphological observations, we evaluated the role of the non-canonical Wnt-5a ligand in oocytes. We found that acute application of Wnt-5a generated changes in endogenous calcium-dependent currents, entry oscillatory current, the membrane's outward current, and induced membrane depolarization. The incubation of oocytes with Wnt-5a caused a reduction of the membrane potential, potassium outward current, and protected the ATP current in the epithelium/theca removed (ETR) model. The oocytes exposed to Wnt-5a showed increased viability and an increase in the percentage of the germinal vesicle breakdown (GVBD), at a higher level than the control with progesterone. Altogether, our results suggest that Wnt-5a modulates different aspects of oocyte structure and generates calcium-dependent endogenous current alteration and GVDB process with a change in membrane potential at different concentrations and times of the exposition. These results help to understand the cellular effect of Wnt-5a and present the use of Xenopus oocytes to explore the mechanism that could impact the activation of Wnt signaling.
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Blood extraction is extremely important for the development of scientific research; however, the existing methods for amphibian´s blood sampling are invasive, mainly leading to the euthanasia of the animal. Therefore, less intrusive methods that allow the obtention of multiple samples from the same individual, are needed as an alternative to the common methods available. Hence, the aim of this study was to propose a minimally invasive method for obtaining blood from the hind leg of Xenopus laevis, that allows continuous sampling without compromising the wellbeing of the organisms. With this method, it was possible to extract blood and plasma from adults and juveniles, and the amount of sample was enough to perform biochemical and molecular assays to assess the viability of the blood. The results also revealed that this method is a convenient alternative to obtain blood without affecting the welfare of the experimental organisms, avoiding the cull of the animals, and the samples are viable for their use in follow up studies.
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Eutanasia Animal , Procedimientos Quirúrgicos Mínimamente Invasivos , Animales , Xenopus laevis , Estudios de Seguimiento , Procedimientos Quirúrgicos Mínimamente Invasivos/veterinariaRESUMEN
Cadmium (Cd) is often detected in the environment due to its wide use in industry; also, NSAIDs are one of the most consumed pharmaceuticals, particularly diclofenac (DCF). Several studies have reported the presence of both contaminants in water bodies at concentrations ranging from ng L-1 to µg L-1; in addition, they have shown that they can induce oxidative stress in aquatic species and disturb signal transduction, cell proliferation, and intercellular communication, which could lead to teratogenesis. Spirulina has been consumed as a dietary supplement; its antioxidant, anti-inflammatory, neuroprotective, and nutritional properties are well documented. This work aimed to evaluate if Spirulina reduces the damage induced by Cd and DCF mixture in Xenopus laevis at early life stages. FETAX assay was carried out: 20 fertilized oocytes were exposed to seven different treatments on triplicate, control, Cd (24.5 µg L-1), DCF (149 µg L-1), Cd + DCF, Cd+DCF+Spirulina (2 mg L-1), Cd+DCF+Spirulina (4 mg L-1), Cd+DCF+Spirulina (10 mg L-1), malformations, mortality, and growth were evaluated after 96 h, also lipid peroxidation, superoxide dismutase and catalase activity were determined after 192 h. Cd increased DCF mortality, Cd and DCF mixture increased the incidence of malformations as well as oxidative damage; on the other hand, the results obtained show that Spirulina can be used to reduce the damage caused by the mixture of Cd and DCF since it promotes growth, reduce mortality, malformations, and oxidative stress in X. laevis.
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Antiinflamatorios no Esteroideos , Spirulina , Animales , Antiinflamatorios no Esteroideos/toxicidad , Spirulina/metabolismo , Xenopus laevis , Cadmio/toxicidad , Diclofenaco/toxicidad , Estrés Oxidativo , Antioxidantes/farmacología , MetalesRESUMEN
Mammals have a limited regenerative capacity, especially of the central nervous system. Consequently, any traumatic injury or neurodegenerative disease results in irreversible damage. An important approach to finding strategies to promote regeneration in mammals has been the study of regenerative organisms like Xenopus, the axolotl, and teleost fish. High-throughput technologies like RNA-Seq and quantitative proteomics are starting to provide valuable insight into the molecular mechanisms that drive nervous system regeneration in these organisms. In this chapter, we present a detailed protocol for performing iTRAQ proteomics that can be applied to the analysis of nervous system samples, using Xenopus laevis as an example. The quantitative proteomics protocol and directions for performing functional enrichment data analyses of gene lists (e.g., differentially abundant proteins from a proteomic study, or any type of high-throughput analysis) are aimed at the general bench biologist and do not require previous programming knowledge.
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Enfermedades Neurodegenerativas , Animales , Proteómica , Regeneración Nerviosa , Sistema Nervioso Central , Análisis de Datos , Xenopus laevis , MamíferosRESUMEN
Xenopus laevis oocytes have been an invaluable tool to discover and explore the molecular mechanisms and characteristics of many proteins, in particular integral membrane proteins. The oocytes were fundamental in many projects designed to identify the cDNA encoding a diversity of membrane proteins including receptors, transporters, channels and pores. In addition to being a powerful tool for cloning, oocytes were later used to experiment with the functional characterization of many of the identified proteins. In this review I present an overview of my personal 30-year experience using Xenopus laevis oocytes and the impact this had on a variety of fields such as arterial blood pressure, neuronal excitability, mineral metabolism and cell volume regulation.
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BACKGROUND: In a high-throughput RNA sequencing analysis, comparing the transcriptional response between Xenopus laevis regenerative and non-regenerative stages to spinal cord injury, cornifelin was found among the most highly differentially expressed genes. Cornifelin is mainly expressed in stratified squamous epithelia, but its expression in the spinal cord and other central nervous structures has only been described during early development. RESULTS: Here, we report cornifelin expression in the spinal cord, retina, and cornea throughout metamorphosis and in the spinal cord after injury. Cornifelin was detected in the grey matter and meninges of the spinal cord from NF-50 to NF-66, with decreased expression in the grey matter during metamorphosis. In the retina, cornifelin was expressed in the ganglion cell layer, the inner and outer nuclear layer, and the outer segment from NF-50 to NF-66. After spinal cord injury, we only observed cornifelin upregulation in NF-66 but no significant changes in NF-50. However, we found cornifelin positive cells in NF-50 meninges closing the spinal cord stumps 1 day after injury and delineating the borders of the spinal cord following the continuity of tissue regeneration in the following days after injury. Instead, in NF-66, cornifelin positive cells were distributed to the ventral side of the spinal cord at 6 days after injury, and at the injury gap at 10 days after injury. CONCLUSIONS: Cornifelin is expressed in the Xenopus laevis spinal cord and eye during metamorphosis and plays a role in the meningeal response to spinal cord injury.
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Traumatismos de la Médula Espinal , Animales , Metamorfosis Biológica/genética , Médula Espinal , Traumatismos de la Médula Espinal/genética , Xenopus laevis/genéticaRESUMEN
While cartilage is an ancient tissue found both in protostomes and deuterostomes, its mineralization evolved more recently, within the vertebrate lineage. SPARC, SPARC-L, and the SCPP members (Secretory Calcium-binding PhosphoProtein genes which evolved from SPARC-L) are major players of dentine and bone mineralization, but their involvement in the emergence of the vertebrate mineralized cartilage remains unclear. We performed in situ hybridization on mineralizing cartilaginous skeletal elements of the frog Xenopus tropicalis (Xt) and the shark Scyliorhinus canicula (Sc) to examine the expression of SPARC (present in both species), SPARC-L (present in Sc only) and the SCPP members (present in Xt only). We show that while mineralizing cartilage expresses SPARC (but not SPARC-L) in Sc, it expresses the SCPP genes (but not SPARC) in Xt, and propose two possible evolutionary scenarios to explain these opposite expression patterns. In spite of these genetic divergences, our data draw the attention on an overlooked and evolutionarily conserved peripheral cartilage subdomain expressing SPARC or the SCPP genes and exhibiting a high propensity to mineralize.
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Spirulina (Arthrospira maxima) has been recognized as a superfood and nutraceutical by its high nutritional value and the benefits of its consumption; it is an important source of lipids, proteins, vitamins, minerals, and antioxidants. It is known that spirulina has positive effects on the toxicity induced by pharmaceuticals and metals. Heavy metals such as cadmium, frequently used in industrial activities, are continuously detected in water bodies and can generate adverse effects on aquatic organisms even at low concentrations. This study aimed to evaluate the protective effect of spirulina (Arthrospira maxima) against the toxic effects induced by cadmium in the early life stages of Xenopus laevis. Twenty Xenopus laevis embryos were exposed to five different treatments on triplicate, control, cadmium (CdCl2 24.5 µg L-1) and three spirulina mixtures Cd + S 1 (24.5 µg L-1 CdCl2 + 2 mg L-1 spirulina), Cd + S 2 (24.5 µg L-1 CdCl2 + 2 mg L-1 spirulina), Cd + S 3 (24.5 µg L-1 CdCl2 + 10 mg L-1 spirulina); after 96 h of exposure: Malformations, mortality and length were evaluated; also, after 192 h, lipid peroxidation (LPX), superoxide dismutase (SOD) and catalase (CAT) were determined. All spirulina treatments decreased mortality from 34 to 50% and reduced malformations on incidence from 36 to 68%. Treatment Cd + S 3 decreased growth inhibition significantly. Spirulina treatment Cd + S 2 decreased lipidic peroxidation and antioxidant activity; these results suggest that spirulina (Arthrospira maxima) can decrease the mortality, frequency of malformations, the severity of malformations, growth inhibition, and oxidative damage induced by cadmium in Xenopus laevis embryos.
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Cloruro de Cadmio/toxicidad , Estrés Oxidativo/efectos de los fármacos , Spirulina , Contaminantes Químicos del Agua/toxicidad , Xenopus laevis , Anomalías Inducidas por Medicamentos/prevención & control , Animales , Catalasa/genética , Catalasa/metabolismo , Embrión no Mamífero/efectos de los fármacos , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Larva/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Non-dystrophic myotonias have been linked to loss-of-function mutations in the ClC-1 chloride channel or gain-of-function mutations in the Nav1.4 sodium channel. Here, we describe a family with members diagnosed with Thomsen's disease. One novel mutation (p.W322*) in CLCN1 and one undescribed mutation (p.R1463H) in SCN4A are segregating in this family. The CLCN1-p.W322* was also found in an unrelated family, in compound heterozygosity with the known CLCN1-p.G355R mutation. One reported mutation, SCN4A-p.T1313M, was found in a third family. Both CLCN1 mutations exhibited loss-of-function: CLCN1-p.W322* probably leads to a non-viable truncated protein; for CLCN1-p.G355R, we predict structural damage, triggering important steric clashes. The SCN4A-p.R1463H produced a positive shift in the steady-state inactivation increasing window currents and a faster recovery from inactivation. These gain-of-function effects are probably due to a disruption of interaction R1463-D1356, which destabilizes the voltage sensor domain (VSD) IV and increases the flexibility of the S4-S5 linker. Finally, modelling suggested that the p.T1313M induces a strong decrease in protein flexibility on the III-IV linker. This study demonstrates that CLCN1-p.W322* and SCN4A-p.R1463H mutations can act alone or in combination as inducers of myotonia. Their co-segregation highlights the necessity for carrying out deep genetic analysis to provide accurate genetic counseling and management of patients.
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Canales de Cloruro/genética , Mutación/genética , Miotonía Congénita/genética , Miotonía/genética , Canal de Sodio Activado por Voltaje NAV1.4/genética , Femenino , Pruebas Genéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Miotonía Congénita/metabolismo , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , LinajeRESUMEN
G protein-activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.
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Secuencias de Aminoácidos , Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Oocitos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animales , Secuencia Conservada , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Humanos , Oocitos/efectos de los fármacos , Filogenia , Unión Proteica , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Amphibians' integument is a multifunctional organ offering protection from the exterior surroundings and facilitating the physiological change of gas, water and salts with the environment, which is a natural biomaterial with multifunctional features. Interspecies comparison of biomechanical characters and microstructure possibly related to them were performed on the integument of three species of amphibians, two anurans(Rana dybowskii and Xenopus laevis) and one urodeles(Ambystoma mexicanum) using tensile testing and morphological characterization. It was found that the integument of Rana dybowskii and Xenopus laevis was covered by polygonal epidermal cells, while the trunk surface of Ambystoma mexicanum presented irregular microstructure with the lack of keratinization. The integument of Rana dybowskii and Xenopus laevis exhibited good performance on stiffness and strength, which showed quite high mean elastic modulus, 931MPa and 1048MPa,respectively.
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Ambystoma mexicanum , Ranidae , Animales , Xenopus laevisRESUMEN
Geoffroea decorticans (chañar) is commonly used for culinary and medicinal purposes in rural communities. The aim of this work was to chemically characterize three Geoffroea decorticans extracts and determine their capacity to modulate the wnt/ß-catenin pathway. This signaling pathway plays a key role in embryonic development but its overactivation leads to cancer cell growth. Phytochemical analysis of extracts showed presence of major classes of phytochemicals. Gas chromatography-mass spectrometry results revealed the presence of acids, esters and furanic compounds. Using Xenopus embryos as in vivo model organisms, we found that the extracts modulated dorso-ventral axis formation and rescued hyperdorsalized phenotypes produced by LiCl treatment. In agreement with these findings, Geoffroea decorticans extracts decreased ß-catenin levels and suppressed the expression of wnt target genes such as xnr3 and chordin, thus demonstrating an inhibitory regulation of the wnt/ß-catenin signaling pathway. All these results support a new role for Geoffroea decorticans fruit derivatives with possible anti-carcinogenic actions.
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Fabaceae/química , Frutas/química , Terapia Molecular Dirigida , Neoplasias/metabolismo , Extractos Vegetales/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Xenopus laevis , beta Catenina/antagonistas & inhibidores , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Cloruro de Litio/farmacología , Masculino , Neoplasias/tratamiento farmacológico , Extractos Vegetales/química , Factor de Crecimiento Transformador beta/genética , Vía de Señalización Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. METHODS: We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP+ cells for RNAseq analysis. RESULTS: In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. CONCLUSIONS: The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.
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Células-Madre Neurales , Traumatismos de la Médula Espinal , Regeneración de la Medula Espinal , Animales , Médula Espinal , Xenopus laevis , Pez CebraRESUMEN
The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.
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Vías Eferentes/fisiología , Sistema de la Línea Lateral/fisiología , Sistema Nervioso Parasimpático/fisiología , Sinapsis/fisiología , Animales , Bungarotoxinas/farmacología , Señalización del Calcio/efectos de los fármacos , Regulación de la Expresión Génica , Células Ciliadas Auditivas/fisiología , Antagonistas Nicotínicos/farmacología , Oocitos , Estimulación Física , Receptores Nicotínicos/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Estricnina/farmacología , Xenopus , Pez CebraRESUMEN
This essay represents a critical analysis of the literary data on various types of waves occurring in the amphibian embryos during gastrulation. A surface contraction wave travels through the presumptive neurectoderm during Mexican axolotl gastrulation. This wave coincides temporally and spatially with involution of the inducing chordomesoderm and with the prospective neural plate. By contrast, there is no similar surface contraction wave during African clawed frog gastrulation. However, the clawed frog displays the waves of DNA synthesis and mitosis in the presumptive neurectoderm during gastrulation, whereas no such waves were discovered in axolotl gastrulae. These sets of experimental data are in accordance with the contemporary concept of considerable ontogenetic diversity of the class Amphibia.
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Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Gástrula/fisiología , Gastrulación/fisiología , Placa Neural/fisiología , Ambystoma mexicanum , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , Gástrula/citología , Gastrulación/genética , Mitosis/genética , Mitosis/fisiología , Placa Neural/citología , Especificidad de la Especie , Xenopus laevisRESUMEN
Feed plays a central role in the physiological development of terrestrial and aquatic animals. Historically, the feeding practice of aquatic research species derived from aquaculture, farmed, or ornamental trades. These diets are highly variable, with limited quality control, and have been typically selected to provide the fastest growth or highest fecundity. These variations of quality and composition of diets may affect animal/colony health and can introduce confounding experimental variables into animal-based studies that impact research reproducibility.
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Acuicultura/métodos , Animales , Animales de Laboratorio , Cefalópodos/fisiología , Modelos Animales , Xenopus/fisiología , Pez Cebra/fisiologíaRESUMEN
The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.
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Nicotiana , Proteínas de Plantas , Secuencia de Aminoácidos , Moduladores del Transporte de Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inhibidores de Proteasas , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
The deregulation of the Wnt/ß-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/ß-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/ß-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs ß-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/ß-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.
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Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.
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Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Cationes Bivalentes/metabolismo , Humanos , Unión Proteica , Dominios ProteicosRESUMEN
BACKGROUND: Lin28 regulates stem cell biology and developmental timing. At the molecular level Lin28 inhibits the biogenesis of the micro RNA let-7 and directly controls the transcription and translation of several genes. In Xenopus, Lin28 overexpression delays metamorphosis and affects the expression of genes of the thyroid hormone (TH) axis. The TH carrier albumin, synthesized by the liver, is down-regulated in limbs and tail after Lin28 overexpression. The molecular mechanisms underlying the interaction between Lin28, let-7, and the hypothalamus-pituitary-thyroid gland (HPT) axis are unknown. RESULTS: We found that precursor and mature forms of let-7 increase during Xenopus metamorphosis. In the liver, lin28b is down-regulated and albumin is up-regulated during metamorphosis. Overexpression of a truncated form of Lin28a (Lin28aΔC), which has been shown not to interact with RNA helicase A to regulate translation, delays metamorphosis, indicating that the translational regulation domain is not required to inhibit the HPT axis. Importantly, both full length Lin28a and Lin28aΔC block the increase of albumin mRNA in the liver independently of changes in TH signaling. CONCLUSIONS: These results suggest that Lin28 delays metamorphosis through regulation of let-7 and that the decrease of the TH carrier albumin is one of the early changes after Lin28 overexpression.