Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver
Analiza interakcije jedarnog glukokortikoidnog receptora i DNK u jetri pacova tokom starenja
Апстракт
Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p LT 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2..., it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.
U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p LT 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet... za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.
Кључне речи:
ageing / liver / glucocorticoid receptor / GRE / EMSAИзвор:
Journal of the Serbian Chemical Society, 2005, 70, 5, 705-712Издавач:
- Serbian Chemical Society
DOI: 10.2298/JSC0505705V
ISSN: 0352-5139
WoS: 000230375900004
Scopus: 2-s2.0-31544483056
Институција/група
IHTMTY - JOUR AU - Vujčić, Miroslava AU - Terzić, Nataša AU - Ristic-Fira, A AU - Kanazir, D AU - Ruzdijic, S PY - 2005 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/204 AB - Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p LT 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. AB - U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p LT 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja. PB - Serbian Chemical Society T2 - Journal of the Serbian Chemical Society T1 - Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver T1 - Analiza interakcije jedarnog glukokortikoidnog receptora i DNK u jetri pacova tokom starenja VL - 70 IS - 5 SP - 705 EP - 712 DO - 10.2298/JSC0505705V ER -
@article{ author = "Vujčić, Miroslava and Terzić, Nataša and Ristic-Fira, A and Kanazir, D and Ruzdijic, S", year = "2005", abstract = "Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p LT 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing., U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p LT 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.", publisher = "Serbian Chemical Society", journal = "Journal of the Serbian Chemical Society", title = "Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver, Analiza interakcije jedarnog glukokortikoidnog receptora i DNK u jetri pacova tokom starenja", volume = "70", number = "5", pages = "705-712", doi = "10.2298/JSC0505705V" }
Vujčić, M., Terzić, N., Ristic-Fira, A., Kanazir, D.,& Ruzdijic, S.. (2005). Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver. in Journal of the Serbian Chemical Society Serbian Chemical Society., 70(5), 705-712. https://doi.org/10.2298/JSC0505705V
Vujčić M, Terzić N, Ristic-Fira A, Kanazir D, Ruzdijic S. Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver. in Journal of the Serbian Chemical Society. 2005;70(5):705-712. doi:10.2298/JSC0505705V .
Vujčić, Miroslava, Terzić, Nataša, Ristic-Fira, A, Kanazir, D, Ruzdijic, S, "Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver" in Journal of the Serbian Chemical Society, 70, no. 5 (2005):705-712, https://doi.org/10.2298/JSC0505705V . .