cytochrome-c-t and phthalic-acid

Di-(2-ethylhexyl) phthalate (DEHP) is one of the most used plasticizers which have contaminated environment widely, and its extensive use causes female reproductive injury. Melatonin has a substantial protective effect against female reproductive toxicity. This study was undertaken to investigate the influence of melatonin on DEHP-induced damage of human granulosa cells (GCs) in vitro and explore the potential mechanisms. Here, we found that melatonin treatment alleviated DEHP-induced human GCs apoptosis and improved mitochondrial function via inhibiting dynamin-related protein 1 (Drp1) mediated mitochondrial fission. Melatonin inhibited the expression, activation and oligomerization of Drp1, which decreased translocation of Drp1 to mitochondria in DEHP-exposed human GCs. Inhibition of mitochondrial fission reduced intracellular reactive oxygen species (ROS) production, sustained mitochondrial membrane potential and decreased cytochrome c release. Further research showed that AMPK-PGC-1α signal pathway was involved in the inhibition of melatonin on Drp1 expression and activation. Melatonin treatment promoted AMPK activation suppressed by DEHP, and activated AMPK recovered the balance of Drp1 phosphorylation at Ser616 and Ser637 sites and enhanced PGC-1α expression. Moreover, PGC-1α could prevent mitochondrial fission by decreasing Drp1 expression directly via binding to its promoter. In contrast, blocking of AMPK or PGC-1α with specific inhibitor negated the protective effects of melatonin on mitochondrial homeostasis and GCs apoptosis. In summary, our results indicated the protective effects of melatonin on improving mitochondrial function and attenuating cells injury in DEHP-exposed human GCs. Melatonin treatment may be a promising therapeutic approach against DEHP-induced reproductive disorder.

Topics: AMP-Activated Protein Kinases; Apoptosis; Cytochromes c; Diethylhexyl Phthalate; Dynamins; Female; Granulosa Cells; Humans; Melatonin; Mitochondrial Dynamics; Phthalic Acids; Plasticizers; Reactive Oxygen Species

Protein folding has emerged as a central problem in biophysics, and the equilibrium folding mechanism of cytochrome c (cyt c) has served as a model system. Unfortunately, the detailed characterization of both the folding process and of any intermediate that might be populated has been limited by the low structural and/or temporal resolution of the available techniques. Here, we report the use of a recently developed technique to study folding that is based on the site-selective incorporation of carbon-deuterium (C-D) bonds and their characterization by IR spectroscopy. Specifically, we synthesize and characterize the protein with deuterated residues spread throughout four structural motifs: (d3)Leu68 in the 60's helix, (d8)Lys72 and (d8)Lys73 in the 70's helix, (d8)Lys79, (d3)Met80, and (d3)Ala83 in the D-loop, and (d3)Leu94, (d3)Leu98, and (d3)Ala101 in the C-terminal helix. The data reveal correlated behavior of the residues within each structural motif, as well as between the residues of the 60's and C-terminal helices and between residues of the 70's helix and D-loop. Residues of the 70's helix and the D-loop are more stable than those within the 60's and C-terminal helices, although the former are more sensitive to added denaturant. The data also suggest that the hydrophobicity of the heme cofactor plays a central role in folding. These results contrast with those from previous H/D exchange studies and suggest that the low denaturant fluctuations observed in the H/D exchange experiments are not similar to those through which the protein actually unfolds. The inherently fast time scale of IR also allows us to characterize the folding intermediate, long thought to be present, but which has proven difficult to characterize by other techniques.

Topics: Cytochromes c; Microscopy, Atomic Force; Phthalic Acids; Potassium Dichromate; Protein Folding; Solutions