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Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Janjanam J, Singh S, Jena MK, Varshney N, Kola S, Kumar S, Kaushik JK, Grover S, Dang AK, Mukesh M, Prakash BS, Mohanty AK - PLoS ONE (2014)

Bottom Line: Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity.The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation.The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Center, National Dairy Research Institute, Karnal, India.

ABSTRACT
Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

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Validation of 2D-DIGE data by quantitative PCR and compared with the 2D-DIGE data.Quantitative PCR analysis was performed for the genes shown in the figure and compared its fold expression change in relation to the 2D-DIGE data. A. The expression profile of proteins at peak and late stage of lactation were represented in comparison to early stage that was normalized to 1. B. The expression profile of proteins of Hy and KF samples were represented in comparison to Ly samples that was normalized to 1.
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pone-0102515-g012: Validation of 2D-DIGE data by quantitative PCR and compared with the 2D-DIGE data.Quantitative PCR analysis was performed for the genes shown in the figure and compared its fold expression change in relation to the 2D-DIGE data. A. The expression profile of proteins at peak and late stage of lactation were represented in comparison to early stage that was normalized to 1. B. The expression profile of proteins of Hy and KF samples were represented in comparison to Ly samples that was normalized to 1.

Mentions: The expression levels of some differentially regulated proteins during lactation were confirmed by western blot and quantitative PCR. Antibodies with bovine cross reactivity were not available for most of these proteins and we performed western blot for annexin A1, actin-related protein 3 homolog (ARP3), phosphoglycerate mutase 1 (PGAM1), lamin B1 and vimentin whose antibodies were available commercially and can cross react with bovine. The western blot analysis of these five proteins corresponds to expression profile of proteomics data (Figure 11). We performed quantitative PCR for some of these proteins but the expression at protein levels were not always correlated to mRNA levels (Figure 12). Expression levels of casein isoforms, macropain, serpin B1 and protein disulfide isomerase did not correlate with its mRNA levels. The results indicate that the differentially expressed proteins during lactation stages and milk yielding variability are regulated at protein level and/or due to post-translational modifications of these proteins.


Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Janjanam J, Singh S, Jena MK, Varshney N, Kola S, Kumar S, Kaushik JK, Grover S, Dang AK, Mukesh M, Prakash BS, Mohanty AK - PLoS ONE (2014)

Validation of 2D-DIGE data by quantitative PCR and compared with the 2D-DIGE data.Quantitative PCR analysis was performed for the genes shown in the figure and compared its fold expression change in relation to the 2D-DIGE data. A. The expression profile of proteins at peak and late stage of lactation were represented in comparison to early stage that was normalized to 1. B. The expression profile of proteins of Hy and KF samples were represented in comparison to Ly samples that was normalized to 1.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4128602&req=5

pone-0102515-g012: Validation of 2D-DIGE data by quantitative PCR and compared with the 2D-DIGE data.Quantitative PCR analysis was performed for the genes shown in the figure and compared its fold expression change in relation to the 2D-DIGE data. A. The expression profile of proteins at peak and late stage of lactation were represented in comparison to early stage that was normalized to 1. B. The expression profile of proteins of Hy and KF samples were represented in comparison to Ly samples that was normalized to 1.
Mentions: The expression levels of some differentially regulated proteins during lactation were confirmed by western blot and quantitative PCR. Antibodies with bovine cross reactivity were not available for most of these proteins and we performed western blot for annexin A1, actin-related protein 3 homolog (ARP3), phosphoglycerate mutase 1 (PGAM1), lamin B1 and vimentin whose antibodies were available commercially and can cross react with bovine. The western blot analysis of these five proteins corresponds to expression profile of proteomics data (Figure 11). We performed quantitative PCR for some of these proteins but the expression at protein levels were not always correlated to mRNA levels (Figure 12). Expression levels of casein isoforms, macropain, serpin B1 and protein disulfide isomerase did not correlate with its mRNA levels. The results indicate that the differentially expressed proteins during lactation stages and milk yielding variability are regulated at protein level and/or due to post-translational modifications of these proteins.

Bottom Line: Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity.The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation.The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Center, National Dairy Research Institute, Karnal, India.

ABSTRACT
Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Show MeSH