iTRAQ-based proteomic analysis of adaptive response in the regenerating limb of the Cynops orientalis newt.

The newt has the powerful capacity to regenerate lost limbs following amputation, and represents an excellent model organism to study regenerative processes. However, the molecular basis of the adaptive response in the regenerating limb of the Chinese fire-bellied newt Cynops orientalis immediately after amputation remains unclear. To better understand the adaptive response immediately after limb amputation at the protein level, we used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS methods to analyze changes in the proteome of the regenerating newt limb that occurred 2 h and 8 h after amputation. We identified 152 proteins with more than 1.5-fold change in expression compared to control. GO annotation analysis classified these proteins into several categories such as signaling, Ca(2+) binding and translocation, transcription and translation, immune response, cell death, cytoskeleton, metabolism, etc. Further ingenuity pathway analysis (IPA) showed that several signaling pathways were significantly changed at 2 h and 8 h after amputation, including EIF2 signaling, acute phase response signaling, tight junction signaling and calcium signaling, suggesting these pathways may be closely related to the adaptive response immediately after limb amputation. This work provides novel insights into understanding the molecular processes related to newt limb regeneration immediately after amputation, and a basis for further study of regenerative medicine.

Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride.

Ionic liquids have recently received considerable attention due to their negligible vapor pressure and substitute for conventional organic solvents. However, their solubility in water and a great deal of literature regarding their toxicity on aquatic organisms have caused much concern in recent years. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C(8)mim][Cl]) on the rat pheochromocytoma (PC12) cell line by cell viability assay and to determine the cytological alterations and damages in PC12 cells after 24h of exposure at the concentrations of 0.07, 0.14, and 0.28 mM of [C(8)mim][Cl]. The results show that [C(8)mim][Cl] inhibits PC12 cell growth and decreases their viabilities in a remarkable dose-dependent manner, and the 24h EC(50) of [C(8)mim][Cl] for PC12 cells is calculated to be around 0.56 mM. Our results also reveal that [C(8)mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca(2+), overproduction of reactive oxygen species, gradually exhausted cellular ATP, mitochondrial permeability transition, and apoptosis in PC12 cells. We suppose that mitochondrial permeability transition and mitochondrial dysfunction maybe the major cytotoxicity mechanism of [C(8)mim]Cl for PC12 cells.