Poster
# 23
Poster |
|
6th Internet World Congress for Biomedical Sciences |
José Manuel Martínez-Martos(1), María Jesús Ramírez-Expósito(2), María Dolores Mayas-Torres(3), María Jesús García-López(4), Isabel Prieto-Gómez(5), Garbiñe Arechaga-Maza(6), Manuel Ramírez-Sánchez(7)
(1)(3)(4)(5)(6)(7)Unit of Physiology. University of Jaén - Jaén. Spain
(2)Unit of Physiology. University of Jaen - Jaén. Spain
|
 |
|
|
|
|
[Cell Biology & Cytology] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
Primary cultures of astrocytes from 21-day-old fetuses were prepared from brain hemispheres as described (9,10). The experimental procedures for animal use and care were in accordance with the European Community Council Directive 86/609/EEC. Fetuses were obtained under sterile conditions from rats and frontal cortex were dissected free of meninges and mechanically dissociated in Dulbecco´s modified Eagle´s medium (DMEM). The cell suspension was vortexed at maximum speed, filtered through a nylon mesh of 80 µm pore size and resuspended in DMEM containing 20% fetal calf serum (FCS) and 1% antibiotics. Every three days in culture, half the medium was replaced until the cultures were confluent (approximately 14 days). After this time, cells were detached and the cell suspension was divided in different tubes; several concentrations of oleic and linoleic fatty acids and cholesterol (1µM, 10 µM and 100 µM) were added. Water-soluble complexes of fatty acids and cholesterol with methyl-ß-cyclodextrin as carrier were used to facilitate the dissolution into the culture medium. The control groups were incubated only with the correspondent amounts of methyl- -cyclodextrin. 50 µL of these cell suspensions were cultured in 96-well plates (Costar) (1.5x105 cells/well) and incubated in a humidified atmosphere of 5% CO2 at 37ºC. The astrocyte cultures contained more than 95% of positive cells for the glial fibrillary acidic protein (GFAP). After 3-4 days of incubation (>90% of confluence) AlaAP, ArgAP, CysAP, LeuAP, TyrAP, and pGluAP activities were analysed in whole cells, using as substrates Alanyl- -naphthylamide (AlaNNap), arginyl- -naphthylamide (ArgNNap), cistinyl- -naphthylamide (CysNNap), Leucyl- -naphthylamide (LeuNNap), tirosyl- -naphthylamide (TyrNNap), and piroglutamyl- -naphthylamide (pGluNNap) in accordance with the modified methods of Greenberg (11), Schwabe and McDonald (12) and Wagner et al. (13). After eliminate the culture medium, the cells were incubated during 30 min at 37ºC with 100 µL of the incubation solution which contain 100 µM of AlaNNap, ArgNNap, CysNNap, LeuNNap, TyrNNap or pGluNNap in artificial cerebrospinal fluid (CSF) (NaCl 116mM, KCl 5.4 mM, MgCl2 0.9 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 10mM) pH 7.2. All the reactions were stopped by addition of 100µl of acetate buffer 0.1M (pH 4.2) containing fast garnet GBC salt and Tween-20. The amount of -naphthylamine released as the result of the enzymatic activity was coupled with the GBC salt appearing a red complex which can be spectrophotometrically determined at 550 nm (14). The proteins were quantified in triplicate by the method of Bradford (15), using BSA as a standard. The specific aminopeptidase activities were expressed as nmol of AlaNNap, ArgNNap, CysNNap, LeuNNap, TyrNNap and pGluNNap hydrolysed per minute per mg of protein, using a standard curve prepared with the latter compound under corresponding assay conditions. The spectrophotometric assay was linear with respect to time of hydrolysis and protein content.
The morphological features of astrocytes in primary culture were checked using phase-contrast microscopy and Giemsa staining.
Statistical analysis.
We used one-way analysis of variance (ANOVA) to analyse differences between groups. Post-hoc comparisons were made using Newman-Keuls test. P-values below 0.05 were considered significant.
|
|
|
|
|
|
|
|
[Cell Biology & Cytology] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
