RESUMEN
RESEARCH QUESTION: Can culture conditions influence the sensitivity of a Mouse Embryo Assay and its potential to detect peroxide-related toxicity in mineral oil samples? DESIGN: Protein type and concentration, embryo density and culture dish design were selected as the variables in the culture system with the potential to influence the assay's sensitivity. Fresh 1-cell mouse embryos were cultured under mineral oil samples with known peroxide concentrations. Protein type (human serum albumin [HSA]â¯+â¯α/ß-Globulins versus HSA versus bovine serum albumin [BSA]), concentration (5 mg/ml versus 0.5 mg/ml), embryo density (25 versus 3 µl/embryo) and culture dish (Petri versus micro-well dish) were adjusted to define the culture conditions with the highest sensitivity. RESULTS: High concentrations of peroxides can be easily detected by current quality control standards. However, for oil samples with a lower concentration of peroxides, supplementing the culture medium with 5 mg/ml of HSAâ¯+â¯alpha/beta-globulins or with HSA resulted in an increased detection of embryo toxicity compared with when BSA was used as the protein supplement. The sensitivity of the assay was greatly reduced when embryos were cultured in groups and when certain micro-well dishes were used. CONCLUSIONS: Current quality control protocols may not be sensitive enough to identify low concentrations of peroxides, which, if undetected, can increase over time and become potentially harmful during gamete and embryo culture. The different parameters established in this study allow the sensitivity of the Mouse Embryo Assays to be optimized to specifically detect peroxides in mineral oil samples prior to their release into the market and their broad use in human IVF.
Asunto(s)
Bioensayo , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/citología , Ratones/embriología , Aceite Mineral/química , Peróxidos/aislamiento & purificación , Animales , Bioensayo/métodos , Bioensayo/normas , Células Cultivadas , Medios de Cultivo/química , Medios de Cultivo/farmacología , Contaminación de Medicamentos , Técnicas de Cultivo de Embriones/normas , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/normas , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Aceite Mineral/farmacología , Peróxidos/toxicidad , Proteínas/fisiología , Control de Calidad , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/normasRESUMEN
Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Saccharomyces cerevisiae/metabolismo , Aequorina/química , Aequorina/metabolismo , Antiportadores/metabolismo , Canales de Calcio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismoRESUMEN
The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.
Asunto(s)
Anticuerpos/farmacología , Leptina/antagonistas & inhibidores , Nanoestructuras/química , Receptores de Leptina/antagonistas & inhibidores , Tejido Adiposo , Animales , Anticuerpos/química , Camélidos del Nuevo Mundo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Hiperinsulinismo , Hígado/anatomía & histología , Ratones , Ratones Endogámicos C57BL , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Tamaño de los Órganos , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Aumento de PesoRESUMEN
A growing body of evidence suggests that mitochondrial dysfunctions play a crucial role in the pathogenesis of various neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting both upper and lower motor neurons. Although ALS is predominantly a sporadic disease, approximately 10% of cases are familial. The most frequent familial form is caused by mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1). A dominant toxic gain of function of mutant SOD1 has been considered as the cause of the disease and mitochondria are thought to be key players in the pathogenesis. However, the exact nature of the link between mutant SOD1 and mitochondrial dysfunctions remains to be established. Here, we briefly review the evidence for mitochondrial dysfunctions in familial ALS and discuss a possible link between mutant SOD1 and mitochondrial dysfunction.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Enfermedades Genéticas Congénitas/enzimología , Mitocondrias/enzimología , Superóxido Dismutasa/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Humanos , Mitocondrias/genética , Mitocondrias/patología , Mutación , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
The neurotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (L-beta-ODAP) is an L-glutamate analogue at alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors in neurons and therefore acts as an excitotoxic substance. Chronic exposure to L-beta-ODAP present in Lathyrus sativus L. (L. sativus) seeds is proposed as the cause of the neurodegenerative disease neurolathyrism, but the mechanism of its action has not been conclusively identified. A key factor in excitotoxic neuronal cell death is a disturbance of the intracellular Ca2+ homeostasis, including changes in the capacity of intracellular Ca2+ stores like the endoplasmic reticulum (ER) or mitochondria. In this study, aequorin and other Ca2+ indicators were used in N2a neuroblastoma cells to investigate alterations of cellular Ca2+ handling after 24 h exposure to L-beta-ODAP. Our data demonstrate increased mitochondrial Ca2+ loading and hyperpolarization of the mitochondrial membrane potential (Psi(m)), which was specific for L-beta-ODAP and not observed with L-glutamate. We conclude that L-beta-ODAP disturbs the ER-mitochondrial Ca2+ signaling axis and thereby renders the cells more vulnerable to its excitotoxic effects that ultimately will lead to cell death.