We report the stability and enzymatic activity of Candida rugosa Lipase (E.C.3.1.1.3) immobilized on gamma-Fe2O3 magnetic nanoparticles. The immobilization strategies were either reacting the enzyme amine group with a nanoparticle surface acetyl, or amine groups. In the former, the enzyme was attached through a C=N bond, while in the latter it was connected using glutaraldehyde. AFM images show an average particle size of 20 +/- 10 nm after deconvolution. The enzymatic activity of the immobilized lipase was determined by following the ester cleavage of p-nitrophenol butyrate. The covalently immobilized enzyme was stabile and reactive over 30 days.
The present studies were performed to determine subtype-specific roles of mitogen-activated protein kinase in chondrogenesis. Erk-1/2 activities, downstream of protein kinase C, decreased as chondrogenesis proceeded, whereas p38 activities, independent of protein kinase C, continuously increased during chondrogenesis. Inhibition of Erk-1/2 with PD98059 enhanced chondrogenesis up to 1.7-fold, whereas inhibition of p38 with SB203580 reduced it to about 30% of the control level. Inhibition of Erk-1/2 or p38 did not affect precartilage condensation. However, cartilage nodule formation was significantly blocked by the inhibition of p38, whereas Erk-1/2 inhibition did not affect it. Modulation of chondrogenesis by the inhibition of Erk-1/2 and p38 was accompanied by altered expression of adhesion molecules in an opposite way. Expression of N-cadherin was reduced as chondrogenesis proceeded. Inhibition of p38 caused sustained expression of N-cadherin, whereas Erk-1/2 inhibition accelerated the reduction of N-cadherin expression. Expression of integrin ␣51 and fibronectin were found to transiently increase during chondrogenesis. Inhibition of p38 caused continuous increase of expression of these molecules, whereas Erk-1/2 inhibition accelerated the decrease of expression of these molecules at a later period of chondrogenesis. Because temporal expression of these adhesion molecules regulates chondrogenesis, the above results indicate that Erk-1/2 and p38 conversely regulate chondrogenesis at post-precartilage condensation stages by modulating expression of adhesion molecules.Formation of cartilage is initiated by differentiation of mesenchymes into chondrocytes. The differentiation process requires proliferation of chondrogenic competent cells that subsequently undergo precartilage condensation (1-3). Precartilage condensation is characterized by cells that are more densely packed in specific regions. This precartilage condensation is an important prerequisite for the initiation of chondrogenesis in mesenchymes in vivo. It becomes evident that several adhesion molecules including cadherins (4-7), integrins (8, 9), and extracellular matrix (ECM) 1 components (9-12) regulate precartilage condensation. Limb mesenchymes undergo spontaneous differentiation to chondrocytes in vitro when cultured at a high seeding density such as micromass culture in the presence of serum. Precartilage condensation similar to that formed in vivo during chondrogenesis occurs in micromass cultures of limb mesenchymes (13). The cellular aggregates that form in early micromass cultures subsequently differentiate into cartilage nodules, which express cartilage-specific molecules such as type II collagen (6, 14-16). However, the signal transduction pathways involved in precartilage condensation and initiated by condensation are not well characterized.We have previously shown that protein kinase C (PKC) positively regulates chondrogenesis of mesenchymes (17,18). PKC is a multigene family composed of 11 known isoforms (19,20). Multiple PKC isoforms su...
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