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- 저자 : Luis A. Barrera (검색결과 2 건)
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Aggrecan catabolism during mesenchymal stromal cell in vitro chondrogenesis
María Lucía Gutiérrez,Johana María Guevara,Olga Yaneth Echeverri,Diego Garzón-Alvarado,Luis Alejandro Barrera 한국통합생물학회 2013 Animal cells and systems Vol.17 No.4
During skeleton formation, mesenchymal cells condense and differentiate into chondrocytes in a process known as chondrogenesis. Glycosaminoglycans (GAGs), main components of aggrecan in the extracellular matrix (ECM),have an important role in this process. An in vitro simplified system has been devised to study chondrogenesis using mesenchymal progenitor cells. Although the capacity of mesenchymal stromal cells to differentiate into the chondrogenic lineage is well established, there is a lack of knowledge with respect to lysosomal enzyme activity during the chondrogenic process. To further understand GAG’s catabolic activities during in vitro chondrogenesis, we evaluated three lysosomal enzymes. Chondrogenic differentiation was demonstrated by Alcian blue positive stain quantified by a grading system using ImageJ. Enzyme activity for N-acetylgalactosamine-6-sulfate-sulfatase during chondrogenic induction decreased significantly with time of culture; b-galactosidase enzyme activity had a similar tendency of temporal activity. On the contrary, b-glucuronidase enzyme activity decreased from the first to second week of induction, but remained the same during the third week of culture. Aggrecan’s immunohistochemistry values for aggregates under chondrogenic induction revealed a similar temporal pattern to that of N-acetylgalactosamine-6-sulfate-sulfatase and b-galactosidase enzyme activity. This work has contributed to the evaluation of enzyme activities associated with GAG degradation, critical component of cartilage ECM. These findings are relevant in understanding the role of enzymes responsible for degradation of molecules predominantly synthesized in the chondrogenic differentiation process. A better understanding of the roles of these enzymes during development could help elucidate further association of deficiencies of these enzymes in skeletal pathologies, primarily chondrodysplasias.
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Edwin David Morales-Álvarez,Claudia Marcela Rivera-Hoyos,Angélica María Baena-Moncada,Patricia Landázuri,Raúl A. Poutou-Piñales,Homero Sáenz-Suárez,Luis A. Barrera,Olga Y. Echeverri-Peña 한국미생물학회 2013 The journal of microbiology Vol.51 No.2
The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold,compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like)emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization,future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.
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