RESUMEN
Neurological and psychiatric side effects accompany the high-dose interferon-alpha (IFNA) therapy. The primary genes responsible for these complications are mostly unknown. Our genome-wide search in mouse and rat genomes for the conservative genes containing IFN-stimulated response elements (ISRE) in their promoters revealed a new potential target gene of IFNA, Grin3α, which encodes the 3A subunit of NMDA receptor. This study aimed to explore the impact of IFNA on the expression of Grin3α and Ifnα genes and neurotransmitters endo/exocytosis in the mouse brain. We administered recombinant human IFN-alpha 2b (rhIFN-α2b) intracranially, and 24 h later, we isolated six brain regions and used the samples for RT-qPCR and western blot analysis. Synaptosomes were isolated from the cortex to analyze endo/exocytosis with acridine orange and L-[14C]glutamate. IFNA induced an increase in Grin3α mRNA and GRIN3A protein, but a decrease in Ifnα mRNA and protein. IFNA did not affect the accumulation and distribution of L-[14C]glutamate and acridine orange between synaptosomes and the extra-synaptosomal space. It caused the more significant acridine orange release activated by NMDA or glutamate than from control mice's synaptosomes. In response to IFNA, the newly discovered association between elevated Grin3α expression and NMDA- and glutamate-evoked neurotransmitters release from synaptosomes implies a new molecular mechanism of IFNA neurotoxicity.
Asunto(s)
Exocitosis/efectos de los fármacos , Interferón alfa-2/toxicidad , Glicoproteínas de Membrana/biosíntesis , N-Metilaspartato/farmacología , Animales , Exocitosis/fisiología , Femenino , Expresión Génica , Humanos , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/toxicidadRESUMEN
The etiology and degree of clinical symptoms of preeclampsia depend on genotypic and phenotypic maternal and trophoblast factors, and elevated levels of plasma homocysteine (Hcy) are one of the pathogenetic factors of preeclampsia. To assess the impact of the folate-related metabolism, we characterized the indices of this metabolism in 40 samples from uncomplicated term placentas and 28 samples from preeclamptic pregnancies by quantifying the total content of folate, methionine (Met), Hcy and related cysteine, and glutathione (GSH) in compliance with the 677 C/T genotype of methylene tetrahydrofolate reductase (MTHFR). The prevalence of MTHFR genotypes was not significantly different between the two groups. The polymorphism of MTHFR was not unambiguously connected with the content of total placental Met, Hcy and related cysteine, and GSH either in uncomplicated or in complicated pregnancies. By contrast, the combination of the heterozygous MTHFR genotype with folate deficiency in the samples from preeclamptic pregnancies was characterized by a statistically significant decrease in the Met content, a trend toward increased Hcy levels and a tight association between metabolically directly and indirectly related compounds, e.g. positive relation between Hcy versus cysteine and folate versus GSH and negative relation between folate versus Hcy and both Hcy and cysteine versus GSH. We demonstrated the expression of cystathionine-ß-synthase (CBS) in human placenta at term by RT-PCR and western blot analysis, for the first time, and confirmed its catalytic activity and the accumulation of cysteine and CBS in placental explants cultivated in the presence of elevated Hcy concentrations. We suggest that disturbance in placental folate-related metabolism may be one of the pathogenetic factors in preeclampsia.
Asunto(s)
Biomarcadores , Ácido Fólico/metabolismo , Placenta/metabolismo , Preeclampsia/metabolismo , Proteínas Gestacionales/metabolismo , Adulto , Biomarcadores/análisis , Biomarcadores/metabolismo , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Modelos Biológicos , Placenta/patología , Polimorfismo Genético , Preeclampsia/diagnóstico , Preeclampsia/genética , Preeclampsia/patología , Embarazo , Proteínas Gestacionales/análisis , Adulto JovenRESUMEN
During the prereplicative period of liver regeneration the changes in the levels of mRNA for tumour necrosis factor-alpha (TNF-alpha) and its receptors were nearly synchronous. The mRNA levels reached their maximum 1-3 h after operation and exceeded the values for intact animals about ten-fold. Lipopolysaccharide stimulation induced an increase in TNF-alpha and TNF receptor production comparable with that occurring during regeneration. Nitric oxide (NO) production in the regenerating liver was determined by electron paramagnetic resonance (EPR) spectroscopy. The first increase in NO production occurred approximately 1 h after partial hepatectomy (PHE). The second and more pronounced peak of NO production was observed about 6 h after PHE when the hepatocytes entered the first cell cycle; it originated mainly from these cells. The consequent minimum of NO synthesis coincided with the maximal rate of DNA synthesis. The third gradual rise of NO production was seen at the transit from the first to the second cell cycle of the hepatocytes and the entrance of the non-parenchymal cells into proliferation. Hepatocytes, Kupffer and endothelial cells were isolated from livers after PHE. They were found to start their main NO production in the described sequence at the times corresponding to their respective entrance into the cell cycle. The maxima of NO synthesis were inversely correlated to the DNA-synthesizing activity of the individual cell type.
Asunto(s)
Regeneración Hepática/fisiología , Hígado/metabolismo , Óxido Nítrico/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , ADN/biosíntesis , Espectroscopía de Resonancia por Spin del Electrón , Hepatectomía , Lipopolisacáridos/farmacología , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de TiempoRESUMEN
Nitric oxide (NO) production by cells of the regenerating liver was estimated from the amount of nitrite accumulated during 24 h in the culture media of hepatocytes, Kupffer cells and sinusoidal endothelial cells isolated at different times after partial hepatectomy (PHE). The time course of NO production was compared with the course of the proliferating activity of the same cells. During the time when liver cells pass through their first cell cycles, hepatocytes were the main producers of NO in the liver. The time-dependent changes of their NO production corresponded to those obtained with the whole liver and were inversely correlated with the DNA-synthesizing activity. The NO production by Kupffer and endothelial cells followed that by hepatocytes in this order; the time displacement between them corresponded to the schedule of their proliferating activity. The NO synthesis in non-parenchymal cells fluctuated in a similar way as in parenchymal cells and was minimal when DNA synthesis was manifest.