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The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats.

Xu YD, Cui JM, Wang Y, Yin LM, Gao CK, Liu YY, Yang YQ - Respir. Res. (2010)

Bottom Line: The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR), which is characterized by acute airway obstruction beginning within a few minutes.Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry.Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yue Yang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

ABSTRACT

Background: The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR), which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma.

Methods: In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism.

Results: In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses.

Conclusions: Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.

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Protein expression profiles in lung and asthmatic lung tissue. (A) Representative 2-D gel images of proteins isolated from normal control (left) and asthmatic rats (right). Total protein extracts were separated on 17-cm linear IPG strips (pH 3-10) in the first dimension followed by 12% SDS-PAGE in the second dimension and visualized by silver staining. (Left) The 25 down-regulated protein spots in asthmatic rats are marked with arrows. (Right) The 19 up-regulated protein spots in asthmatic rats are marked with arrows. The numbers correspond to the spot identification numbers listed in Table 1. The molecular weight standards and the pH range are shown at the left and the bottom of the gels, respectively. (B) Cropped 2-DE gel images of the S100A8 and RhoGDI2 protein spots. The selected area was symmetrically boxed, and the arrows indicate each protein spot or its theoretical location. (C) The expression profile of 28 most significantly changed proteins that were detected more than three times. The upper portion shows the 17 proteins down-regulated in asthmatic lung tissue, and the lower portion shows the 11 proteins that were up-regulated. Each spot volume was quantified from the intensity of the spots using PDQuest software. The bars represent the mean ± SD from three replicated 2-DE gels. The information for each altered spot is reported in Table 1.
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Figure 3: Protein expression profiles in lung and asthmatic lung tissue. (A) Representative 2-D gel images of proteins isolated from normal control (left) and asthmatic rats (right). Total protein extracts were separated on 17-cm linear IPG strips (pH 3-10) in the first dimension followed by 12% SDS-PAGE in the second dimension and visualized by silver staining. (Left) The 25 down-regulated protein spots in asthmatic rats are marked with arrows. (Right) The 19 up-regulated protein spots in asthmatic rats are marked with arrows. The numbers correspond to the spot identification numbers listed in Table 1. The molecular weight standards and the pH range are shown at the left and the bottom of the gels, respectively. (B) Cropped 2-DE gel images of the S100A8 and RhoGDI2 protein spots. The selected area was symmetrically boxed, and the arrows indicate each protein spot or its theoretical location. (C) The expression profile of 28 most significantly changed proteins that were detected more than three times. The upper portion shows the 17 proteins down-regulated in asthmatic lung tissue, and the lower portion shows the 11 proteins that were up-regulated. Each spot volume was quantified from the intensity of the spots using PDQuest software. The bars represent the mean ± SD from three replicated 2-DE gels. The information for each altered spot is reported in Table 1.

Mentions: The protein in the sample from each group was measured at least three times and verified to ensure that the same protein patterns were obtained. In general, we detected approximately 550-650 protein spots in each 2-DE gel and observed a high rate of overlap (>90%) in the parallel gels. In total, 44 protein spots showed consistently differential expression between the asthmatic group and the control in all three parallel experiments, with 25 down-regulated spots and 19 up-regulated spots (Figure 3A). After identification via mass spectrometry, 42 protein spots corresponding to 36 unique proteins were successfully identified. Of the unique proteins, 21 were down-regulated and 16 were up-regulated in the asthmatic rats (Table 1, Figure 3C). For the tubulin alpha-1A chain, both up- and down-regulated expression patterns were observed, possibly reflecting differential post-translational modification of the same protein.


The early asthmatic response is associated with glycolysis, calcium binding and mitochondria activity as revealed by proteomic analysis in rats.

Xu YD, Cui JM, Wang Y, Yin LM, Gao CK, Liu YY, Yang YQ - Respir. Res. (2010)

Protein expression profiles in lung and asthmatic lung tissue. (A) Representative 2-D gel images of proteins isolated from normal control (left) and asthmatic rats (right). Total protein extracts were separated on 17-cm linear IPG strips (pH 3-10) in the first dimension followed by 12% SDS-PAGE in the second dimension and visualized by silver staining. (Left) The 25 down-regulated protein spots in asthmatic rats are marked with arrows. (Right) The 19 up-regulated protein spots in asthmatic rats are marked with arrows. The numbers correspond to the spot identification numbers listed in Table 1. The molecular weight standards and the pH range are shown at the left and the bottom of the gels, respectively. (B) Cropped 2-DE gel images of the S100A8 and RhoGDI2 protein spots. The selected area was symmetrically boxed, and the arrows indicate each protein spot or its theoretical location. (C) The expression profile of 28 most significantly changed proteins that were detected more than three times. The upper portion shows the 17 proteins down-regulated in asthmatic lung tissue, and the lower portion shows the 11 proteins that were up-regulated. Each spot volume was quantified from the intensity of the spots using PDQuest software. The bars represent the mean ± SD from three replicated 2-DE gels. The information for each altered spot is reported in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2925830&req=5

Figure 3: Protein expression profiles in lung and asthmatic lung tissue. (A) Representative 2-D gel images of proteins isolated from normal control (left) and asthmatic rats (right). Total protein extracts were separated on 17-cm linear IPG strips (pH 3-10) in the first dimension followed by 12% SDS-PAGE in the second dimension and visualized by silver staining. (Left) The 25 down-regulated protein spots in asthmatic rats are marked with arrows. (Right) The 19 up-regulated protein spots in asthmatic rats are marked with arrows. The numbers correspond to the spot identification numbers listed in Table 1. The molecular weight standards and the pH range are shown at the left and the bottom of the gels, respectively. (B) Cropped 2-DE gel images of the S100A8 and RhoGDI2 protein spots. The selected area was symmetrically boxed, and the arrows indicate each protein spot or its theoretical location. (C) The expression profile of 28 most significantly changed proteins that were detected more than three times. The upper portion shows the 17 proteins down-regulated in asthmatic lung tissue, and the lower portion shows the 11 proteins that were up-regulated. Each spot volume was quantified from the intensity of the spots using PDQuest software. The bars represent the mean ± SD from three replicated 2-DE gels. The information for each altered spot is reported in Table 1.
Mentions: The protein in the sample from each group was measured at least three times and verified to ensure that the same protein patterns were obtained. In general, we detected approximately 550-650 protein spots in each 2-DE gel and observed a high rate of overlap (>90%) in the parallel gels. In total, 44 protein spots showed consistently differential expression between the asthmatic group and the control in all three parallel experiments, with 25 down-regulated spots and 19 up-regulated spots (Figure 3A). After identification via mass spectrometry, 42 protein spots corresponding to 36 unique proteins were successfully identified. Of the unique proteins, 21 were down-regulated and 16 were up-regulated in the asthmatic rats (Table 1, Figure 3C). For the tubulin alpha-1A chain, both up- and down-regulated expression patterns were observed, possibly reflecting differential post-translational modification of the same protein.

Bottom Line: The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR), which is characterized by acute airway obstruction beginning within a few minutes.Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry.Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Yue Yang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

ABSTRACT

Background: The inhalation of allergens by allergic asthmatics results in the early asthmatic response (EAR), which is characterized by acute airway obstruction beginning within a few minutes. The EAR is the earliest indicator of the pathological progression of allergic asthma. Because the molecular mechanism underlying the EAR is not fully defined, this study will contribute to a better understanding of asthma.

Methods: In order to gain insight into the molecular basis of the EAR, we examined changes in protein expression patterns in the lung tissue of asthmatic rats during the EAR using 2-DE/MS-based proteomic techniques. Bioinformatic analysis of the proteomic data was then performed using PPI Spider and KEGG Spider to investigate the underlying molecular mechanism.

Results: In total, 44 differentially expressed protein spots were detected in the 2-DE gels. Of these 44 protein spots, 42 corresponded to 36 unique proteins successfully identified using mass spectrometry. During subsequent bioinformatic analysis, the gene ontology classification, the protein-protein interaction networking and the biological pathway exploration demonstrated that the identified proteins were mainly involved in glycolysis, calcium binding and mitochondrial activity. Using western blot and semi-quantitative RT-PCR, we confirmed the changes in expression of five selected proteins, which further supports our proteomic and bioinformatic analyses.

Conclusions: Our results reveal that the allergen-induced EAR in asthmatic rats is associated with glycolysis, calcium binding and mitochondrial activity, which could establish a functional network in which calcium binding may play a central role in promoting the progression of asthma.

Show MeSH
Related in: MedlinePlus