Tumour necrosis factor (TNF)-a induces a transient increase in N-octanoylsphingosine (C8-ceramide) which has been postulated as an intracellular mediator in TNF-a signalling. We tested the ability of C8-ceramide to reproduce the TNF-a-mediated interference with endothelial cell proliferation and DNA synthesis. TNF-a (10 ng´mL 21 ) and C8-ceramide (20 mm) inhibited the incorporation of [ 3 H]thymidine into DNA and led to an accumulation of cells in the G 1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF-a and C8-ceramide induced increased expression of p53. Treatment with TNF-a or C8-ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF-a prevented RB phosphorylation whereas C8-ceramide did not interfere with this process, suggesting that it follows a ceramide-independent pathway.Keywords: ceramide; endothelial cells; p53; RB protein; TNF-a.N-octanoylsphingosine (C8-ceramide) was initially recognized as a second mediator of vitamin D3 action in HL-60 human leukaemia cells [1]. This lipid can be generated by de novo synthesis or from the hydrolysis of sphingomyelin catalysed by a neutral sphingomyelinase that is activated by cytokines such as tumour necrosis factor (TNF)-a, interleukin-1b, interferon-g and FAS ligand [2±4]. This cytokine-dependent increase in ceramide content lead to the theory that it is an intracellular mediator for cytokine action [2,3,5]. This hypothesis is supported by additional experimental results: (a) TNF-a activates a ceramide dependent protein kinase (CAPK) [6,7] The TNF-a-mediated interference of cellular proliferation in different human tumour cell lines is associated with apoptotic cell death, making it difficult to separate cytostasis from cytotoxicity [5,11,14,15]. TNF-a also interferes with the proliferation of primary human endothelial cells. This effect is associated with an accumulation of cells in the G 1 phase of the cell cycle, a decrease in DNA synthesis and the retention of the tumour suppressor retinoblastoma gene product (RB) in its active (underphosphorylated) state [15±18].In the anti-proliferative effect induced by TNF-a on primary human endothelial cells the participation of RB has been implicated [15], but it is unknown if other tumour suppressors are required. RB and p53 are important control elements of the cell cycle and in particular of the transition from the G 1 to the S phase [19±21]. In their active state, both RB and p53 prevent the initiation of the S phase, leading to the accumulation of cells in G 1 . RB acts as a default inhibitor of the cell cycle progression, whose function is determined by alternating states of under-and hyper-phosphorylation. In its under-phosphorylated state, RB mediates G 1 cell cycle arrest, explained in part by its ability to sequester the elongation factor E2F [21]. Unlike RB t...
A colloidal synthesis’ proof-of-concept based on the Bligh–Dyer emulsion inversion method was designed for integrating into lipid nanoparticles (LNPs) cell-permeating DNA antisense oligonucleotides (ASOs), also known as GapmeRs (GRs), for mRNA interference. The GR@LNPs were formulated to target brain border-associated macrophages (BAMs) as a central nervous system (CNS) therapy platform for silencing neuroinflammation-related genes. We specifically aim at inhibiting the expression of the gene encoding for lipocalin-type prostaglandin D synthase (L-PGDS), an anti-inflammatory enzyme expressed in BAMs, whose level of expression is altered in neuropsychopathologies such as depression and schizophrenia. The GR@LNPs are expected to demonstrate a bio-orthogonal genetic activity reacting with L-PGDS gene transcripts inside the living system without interfering with other genetic or biochemical circuitries. To facilitate selective BAM phagocytosis and avoid subsidiary absorption by other cells, they were functionalized with a mannosylated lipid as a specific MAN ligand for the mannose receptor presented by the macrophage surface. The GR@LNPs showed a high GR-packing density in a compact multilamellar configuration as structurally characterized by light scattering, zeta potential, and transmission electronic microscopy. As a preliminary biological evaluation of the mannosylated GR@LNP nanovectors into specifically targeted BAMs, we detected in vivo gene interference after brain delivery by intracerebroventricular injection (ICV) in Wistar rats subjected to gene therapy protocol. The results pave the way towards novel gene therapy platforms for advanced treatment of neuroinflammation-related pathologies with ASO@LNP nanovectors.
En esta investigación, analicé las bondades que ofrece un aula virtual en el aprendizaje de los estudiantes del bachillerato general. Para la recolección de datos se utilizó encuestas y entrevistas, para lo cual realicé una entrevista a los directivos del colegio, encuesté a 10 docentes, además a 112 estudiantes, teniendo como resultado una mejora en el aprendizaje. Esta investigación propone analizar el impacto del aula virtual en el proceso de aprendizaje en los estudiantes del bachillerato general, para el diseño del entorno virtual de aprendizaje basado en la plataforma Moodle. Analizando los resultados, se establece que, el estudiante debe hacer uso del aula virtual para adquirir nuevos conocimientos para aumentar su rendimiento académico, debido a que esta herramienta tecnológica ayuda a realizar trabajos autónomos enviados por los docentes para complementar las tareas diarias. Utilizamos la plataforma Moodle para el diseño del aula virtual, la cual permite: Distribuir información, intercambiar ideas, experiencias, aplicar, experimentar lo aprendido, evaluar los conocimientos, además, mantener tanto la seguridad como confiabilidad en el Sistema. También se concluye que haciendo uso del aula virtual el estudiante podrá construir su propio conocimiento teniendo como base conocimientos previos, esto gracias al uso sencillo de la tecnología y aplicaciones informáticas.
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