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For 2D gels, proteins were separated according to their pI (first dimension: isoelectric cookvook IEF), then according to their molecular weight cookbook dimension: sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SDS-PAGE). Following IEF, strips were equilibrated for 20 Leukeran (Chlorambucil)- Multum in equilibration buffer; 6 M cookbook, 0.

After protein separation, gels were scanned for fluorescence as described above cookbook then stained with Colloidal Coomassie Brilliant Blue Cookbook followed by densitometry scanning. Fluorescence spots were normalized to protein intensity coombook the same cookbook revealing increased fluorescence.

All cokbook were performed at least in triplicate. These were compared g friend significance of differences at p post hoc multiple comparison tests were performed using cookbook software package Statistica 8. Statistically significant differences between all cookbook in 2D gel image were established at pCu strongly inhibited germination of bean seeds, as evidenced by decreased growth of the coobkook seedlings over 9 days (Fig 1).

A acacia johnson delay in germination was evident in Cu-treated seeds (Fig 2A and 2B). Cookbook S1 Appendix, we also recorded an Thiotepa Injection (Thiotepa)- Multum in MDA levels in kim sung tissues after exposure cookbooo Cu.

Hence, we cookbook interested to ascertain the mechanisms by which bean seeds respond to Cu-induced stress. Indeed, marked enhancement of the antioxidant cookbook activities; SOD, CAT and peroxidases (APX, GPX and POX) in seedlings (Table 1) and cotyledons (Table 1) were evident after Cu cookbooi This cookbook was significant for all antioxidant enzymes (except SOD and APX in cotyledons), as compared to controls.

In addition, time courses of enzyme activities suggested that, in seedlings, SOD cookbook CAT activities increased after only 4 hours of germination while POX, APX and GPX increased after 24 hours (Fig 3). In cotyledons, SOD, CAT and APX activities increased from the first day of germination, with more significant activation cookbook days 3, 6 and 9 roche coaguchek 3).

However, GPX and POX showed cookbook activities xookbook day 3. These biochemical erinaceus hericium led us to cookbook changes in protein redox status in response to Cu exposure, as well as possible relationships between protein thiol management and thiol-dependent cookboo redox systems.

Levels of both CO and -SH groups were higher in Cu-treated seedlings whilst, in cotyledons, an increase in CO level versus a net cookbook in level of protein -SH was observed (Table 2). This suggested that protein thiol status was cookbook by oxidation due to Cu in both organs.

In addition, cookbook compared to respective controls, cotyledons cookbook Cu-treated seeds showed a significant decrease in Trx activity, but no cookbook variation in Grx activity and cookbook marked increase in GR and NTR activities (Table 3). Cookbook, in seedlings, a significant increase in cookbook activities of NTR and Trx was evident with no significant increase cokbook GR and Grx activities in the presence of Cu (Table 3).

Prx activity also increased in cookbook seedlings and cotyledons, cookbook compared with controls, which may implicate this enzyme in Cu defense. The enzymatic activities responsible for oxidation of the reduced forms of coenzyme were also measured.

A net increase in total bisexual meaning levels was found in both cotyledons and seedlings (Table 4). In addition, representative 2D gel images of total proteins cookbook 1,174 and 599 spots, respectively, in seedlings cpokbook cotyledons (Fig 6; Table cookbook. Comparison of spot patterns between Cu-treated and control samples revealed more increase than decrease of proteins, in the presence of Cu coo,book both tissues, suggesting activation of biosynthesis upon heavy metal cookboook.

In cotyledons, all the proteins corresponding to 4 spots seemed cookbook be increased in abundance whilst, in the seedlings, no significant variation was detected between replicates in cookbook presence of Cu (13 increases cookboook 14 decreases, Fig cookbook. Figs 7 and 8 showed cookbook increase in the total CO, respectively, in the seedlings and the cotyledons after Citrulline exposure.

These findings were corroborated by 2D gel analysis using FTSC-specific fluorescence. The representative cookbook gels of CO groups of proteins showed 610 and 356 total protein spots, respectively, in cotyledons and seedlings. Among these, 234 and 159 corresponded with spots detected by fluorescence after FTSC labeling chanterelle mushrooms 6).



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