RESUMO
Cosmetics Europe, The Personal Care Association (known as Colipa before 2012), conducted a program of technology transfer and within/between laboratory reproducibility of MatTek Corporation's EpiOcular™ Eye Irritation Test (EIT) as one of the two human reconstructed tissue test methods. This EIT EpiOcular™ used a single exposure period for each chemical and a prediction model based on a cut-off in relative survival [ ≤60%=irritant (I) (GHS categories 2 and 1); >60%=no classification (NC)]. Test substance single exposure time was 30 min with a 2-h post-exposure incubation for liquids and 90 min with an 18-h post-exposure incubation for solids. Tissue viability was determined by tetrazolium dye (MTT) reduction. Combinations of 20 coded chemicals were tested in 7 laboratories. Standardized laboratory documentation was used by all laboratories. Twenty liquids (11 NC/9 I) plus 5 solids (3 NC/2 I) were selected so that both exposure regimens could be assessed. Concurrent positive (methyl acetate) and negative (water) controls were tested in each trial. In all, 298 independent trials were performed and demonstrated 99.7% agreement in prediction (NC/I) across the laboratories. Coefficients of variation for the% survival for tissues from each treatment group across laboratories were generally low. This protocol has entered in 2010 the experimental phase of a formal ECVAM validation program.
Assuntos
Olho/efeitos dos fármacos , Irritantes/toxicidade , Testes de Toxicidade Aguda/métodos , Alternativas aos Testes com Animais , Comportamento Cooperativo , Europa (Continente) , Humanos , Técnicas In Vitro , Laboratórios , Modelos Biológicos , Reprodutibilidade dos Testes , Transferência de Tecnologia , Estados UnidosRESUMO
Megakaryocytes give rise to platelets that are essential for thrombosis and hemostasis. During development, megakaryocytes undergo an endomitotic cell cycle by which they skip late anaphase and cytokinesis to yield high ploidy cells. This process is regulated by the c-Mpl receptor ligand. In the current study we used differential display PCR as well as degenerate cloning of kinases to identify part of the program of genes regulated during Mpl ligand-induced differentiation. Several of the induced genes were identified as encoding metabolic proteins as carnitine palmitolytransferase, while other altered genes were identified as encoding kinases. Of these, AIM-1 kinase mRNA was severely downregulated by Mpl ligand at the onset of polyploidy in megakaryocytes. This effect was not related to message stability, but rather to a change in transcriptional rate. These data point to the potential importance of the transcriptional regulation of the AIM-1 gene for promoting megakaryocyte polyploidization.
Assuntos
Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trombopoetina/metabolismo , Animais , Aurora Quinases , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombopoetina/farmacologiaRESUMO
Aspergillus nidulans nimA gene encodes a serine/threonine protein kinase (NIMA) whose activity is essential for mitotic entry and chromatin condensation. Both the activity and the abundance of NIMA protein increase at the G2/M transition of the fungal cell cycle. In this study, we report the effects elicited by ectopic expression of nimA on polyploidization in a mouse megakaryocytic line, Y10, which is undergoing an endomitotic cell cycle. A pool of Y10 stable transfectants that have been induced to express nimA displayed a decrease in cell number and an elevated DNA content per cell. NIMA also dramatically enhanced the activity of phorbal 12-myristate 13-acetate toward polyploidization. Analysis of individual nimA transfectants revealed that the DNA content per cell rose in cells expressing high levels of nimA and that the level of cyclin B was reduced as compared to the mock-transfected cells. These effects observed in polyploidizing megakaryocytes are in contrast to those found in A. nidulans and HeLa cells, in which induced nimA expression caused abnormal chromatin condensation and cell cycle arrest. We conclude that high-level expression of nimA in cells programmed to undergo endomitosis could potentiate polyploidization. The challenge now resides in the isolation of the authentic megakaryocyte counterpart of the fungal nimA.
