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Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries
Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatin...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393235/ https://www.ncbi.nlm.nih.gov/pubmed/25859655 http://dx.doi.org/10.1371/journal.pone.0123852 |
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author | Chen, Shanyan Meng, Fanjun Chen, Zhenzhou Tomlinson, Brittany N. Wesley, Jennifer M. Sun, Grace Y. Whaley-Connell, Adam T. Sowers, James R. Cui, Jiankun Gu, Zezong |
author_facet | Chen, Shanyan Meng, Fanjun Chen, Zhenzhou Tomlinson, Brittany N. Wesley, Jennifer M. Sun, Grace Y. Whaley-Connell, Adam T. Sowers, James R. Cui, Jiankun Gu, Zezong |
author_sort | Chen, Shanyan |
collection | PubMed |
description | Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions. |
format | Online Article Text |
id | pubmed-4393235 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-43932352015-04-21 Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries Chen, Shanyan Meng, Fanjun Chen, Zhenzhou Tomlinson, Brittany N. Wesley, Jennifer M. Sun, Grace Y. Whaley-Connell, Adam T. Sowers, James R. Cui, Jiankun Gu, Zezong PLoS One Research Article Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions. Public Library of Science 2015-04-10 /pmc/articles/PMC4393235/ /pubmed/25859655 http://dx.doi.org/10.1371/journal.pone.0123852 Text en © 2015 Chen et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Chen, Shanyan Meng, Fanjun Chen, Zhenzhou Tomlinson, Brittany N. Wesley, Jennifer M. Sun, Grace Y. Whaley-Connell, Adam T. Sowers, James R. Cui, Jiankun Gu, Zezong Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries |
title | Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries |
title_full | Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries |
title_fullStr | Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries |
title_full_unstemmed | Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries |
title_short | Two-Dimensional Zymography Differentiates Gelatinase Isoforms in Stimulated Microglial Cells and in Brain Tissues of Acute Brain Injuries |
title_sort | two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393235/ https://www.ncbi.nlm.nih.gov/pubmed/25859655 http://dx.doi.org/10.1371/journal.pone.0123852 |
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