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Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes
This study was performed to investigate the transduction of a full-length superoxide dismutase (SOD) protein fused to transactivator of transcription (Tat) into human chondrocytes, and to determine the regulatory function of transduced Tat-SOD in the inflammatory cytokine induced catabolic pathway....
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2006
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1779403/ https://www.ncbi.nlm.nih.gov/pubmed/16792821 http://dx.doi.org/10.1186/ar1972 |
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author | Kim, Hyun Ah Won Kim, Dae Park, Jinseu Choi, Soo Young |
author_facet | Kim, Hyun Ah Won Kim, Dae Park, Jinseu Choi, Soo Young |
author_sort | Kim, Hyun Ah |
collection | PubMed |
description | This study was performed to investigate the transduction of a full-length superoxide dismutase (SOD) protein fused to transactivator of transcription (Tat) into human chondrocytes, and to determine the regulatory function of transduced Tat-SOD in the inflammatory cytokine induced catabolic pathway. The pTat-SOD expression vector was constructed to express the basic domain of HIV-1 Tat as a fusion protein with Cu, Zn-SOD. We also purified histidine-tagged SOD without an HIV-1 Tat and Tat-GFP as control proteins. Cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were cultured in both a monolayer and an explant. For the transduction of fusion proteins, cells/explants were treated with a variety of concentrations of fusion proteins. The transduced protein was detected by fluorescein labeling, western blotting and SOD activity assay. Effects of transduced Tat-SOD on the regulation of IL-1 induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression was assessed by the Griess reaction and reverse transcriptase PCR, respectively. Tat-SOD was successfully delivered into both the monolayer and explant cultured chondrocytes, whereas the control SOD was not. The intracellular transduction of Tat-SOD into cultured chondrocytes was detected after 1 hours, and the amount of transduced protein did not change significantly after further incubation. SOD enzyme activity increased in a dose-dependent manner. NO production and iNOS mRNA expression, in response to IL-1 stimulation, was significantly down-regulated by pretreatment with Tat-SOD fusion proteins. This study shows that protein delivery employing the Tat-protein transduction domain is feasible as a therapeutic modality to regulate catabolic processes in cartilage. Construction of additional Tat-fusion proteins that can regulate cartilage metabolism favorably and application of this technology in in vivo models of arthritis are the subjects of future studies. |
format | Text |
id | pubmed-1779403 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-17794032007-01-19 Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes Kim, Hyun Ah Won Kim, Dae Park, Jinseu Choi, Soo Young Arthritis Res Ther Research Article This study was performed to investigate the transduction of a full-length superoxide dismutase (SOD) protein fused to transactivator of transcription (Tat) into human chondrocytes, and to determine the regulatory function of transduced Tat-SOD in the inflammatory cytokine induced catabolic pathway. The pTat-SOD expression vector was constructed to express the basic domain of HIV-1 Tat as a fusion protein with Cu, Zn-SOD. We also purified histidine-tagged SOD without an HIV-1 Tat and Tat-GFP as control proteins. Cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were cultured in both a monolayer and an explant. For the transduction of fusion proteins, cells/explants were treated with a variety of concentrations of fusion proteins. The transduced protein was detected by fluorescein labeling, western blotting and SOD activity assay. Effects of transduced Tat-SOD on the regulation of IL-1 induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression was assessed by the Griess reaction and reverse transcriptase PCR, respectively. Tat-SOD was successfully delivered into both the monolayer and explant cultured chondrocytes, whereas the control SOD was not. The intracellular transduction of Tat-SOD into cultured chondrocytes was detected after 1 hours, and the amount of transduced protein did not change significantly after further incubation. SOD enzyme activity increased in a dose-dependent manner. NO production and iNOS mRNA expression, in response to IL-1 stimulation, was significantly down-regulated by pretreatment with Tat-SOD fusion proteins. This study shows that protein delivery employing the Tat-protein transduction domain is feasible as a therapeutic modality to regulate catabolic processes in cartilage. Construction of additional Tat-fusion proteins that can regulate cartilage metabolism favorably and application of this technology in in vivo models of arthritis are the subjects of future studies. BioMed Central 2006 2006-06-22 /pmc/articles/PMC1779403/ /pubmed/16792821 http://dx.doi.org/10.1186/ar1972 Text en Copyright © 2006 Kim et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Kim, Hyun Ah Won Kim, Dae Park, Jinseu Choi, Soo Young Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes |
title | Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes |
title_full | Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes |
title_fullStr | Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes |
title_full_unstemmed | Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes |
title_short | Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes |
title_sort | transduction of cu, zn-superoxide dismutase mediated by an hiv-1 tat protein basic domain into human chondrocytes |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1779403/ https://www.ncbi.nlm.nih.gov/pubmed/16792821 http://dx.doi.org/10.1186/ar1972 |
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