ABSTRACT
The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.(AU)
O objetivo deste estudo foi avaliar o efeito direto de concentrações de cafeína sobre a viabilidade, atividade de síntese e expressão gênica em culturas de condrócitos de ratos. As cartilagens dos fêmures e tíbias de 15 ratos Wistar com três dias foram extraídas para isolamento de condrócitos. Os condrócitos foram cultivados em meio condrogênico (controle) ou em meio acrescido de diferentes concentrações de cafeína (0,5, 1,0, 2,0mM). Foram avaliadas a viabilidade celular, a atividade da fosfatase alcalina e a síntese de colágeno por ensaios colorimétricos aos sete, 14 e 21 dias. Condrócitos cultivados sob lamínulas foram corados pela hematoxilina e eosina, para se determinar a porcentagem de células/campo, e pelo PAS, safranina O, alcian Blue, para se determinar a porcentagem de matriz condrogênica/campo aos 21 dias. Foi avaliada a expressão de transcriptos gênicos para Sox-9, Runx-2, agrecano, colágeno-II e fosfatase alcalina por qRT-PCR, aos 21 dias. As médias foram comparadas pelo Student-Newman-Keuls. A cafeína reduziu significativamente o MTT em cristais de formazan, a porcentagem de células/campo, a síntese de colágeno, a atividade da fosfatase alcalina e a síntese de matriz condrogênica PAS+, safranina O+, alcian blue+ e expressão de Sox-9 e colágeno-II. Conclui-se que a cafeína, nas concentrações de 0,5, 1,0, 2,0mM, apresenta efeito inibidor direto sobre a condrogênese em culturas de condrócitos de ratos.(AU)
Subject(s)
Animals , Female , Rats , Caffeine , Cartilage, Articular/drug effects , Chondrocytes/drug effects , Chondrogenesis/drug effectsABSTRACT
The removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl3 or ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.
ABSTRACT
A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.
Subject(s)
Staining and Labeling/methods , Starch/metabolism , Fabaceae/metabolism , Microscopy, Fluorescence , Plants, Medicinal , Seeds/metabolismABSTRACT
Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the alpha type. Starch is degraded slowly in the first days; during this time, alpha- and beta-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in alpha-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three alpha-amylases, and one beta-amylase. Based on their heat lability or heat stability, two sets of alpha-amylase seem to exist in lentil cotyledons.
