alpha-Calcitonin gene-related peptide inhibits autophagy and calpain systems and maintains the stability of neuromuscular junction in denervated muscles.

Objective: Although it is well established that a-calcitonin gene-related peptide (CGRP) stabilizes muscle-type cholinergic receptors nicotinic subunits (AChR), the underlying mechanism by which this neuropeptide regulates muscle protein metabolism and neuromuscular junction (NMJ) morphology is unclear.Methods: To elucidate the mechanisms how CGRP controls NMJ stability in denervated mice skeletal muscles, we carried out physiological, pharmacological, and molecular analyses of atrophic muscles induced by sciatic nerve transection.Results: Here, we report that CGRP treatment in vivo abrogated the deleterious effects on NMJ upon denervation (DEN), an effect that was associated with suppression of skeletal muscle proteolysis, but not stimulation of protein synthesis. CGRP also blocked the DEN-induced increase in endocytic AChR vesicles and the elevation of autophagosomes per NMJ area. The treatment of denervated animals with rapamycin blocked the stimulatory effects of CGRP on mTORC1 and its inhibitory actions on autophagic flux and NMJ degeneration. Furthermore, CGRP inhibited the DEN-induced hyperactivation of Ca2+-dependent proteolysis, a degradative system that has been shown to destabilize NMJ. Consistently, calpain was found to be activated by cholinergic stimulation in myotubes leading to the dispersal of AChR clusters, an effect that was abolished by CGRP.Conclusion: Taken together, these data suggest that the inhibitory effect of CGRP on autophagy and calpain may represent an important mechanism for the preservation of synapse morphology when degradative machinery is exacerbated upon denervation conditions

Partial purification of crude lipase extract from Yarrowia lipolytica: Precipitation, aqueous two-phase systems (ATPS), and immobilization methods

Efforts have been concentrated on developing alternative methods of enzyme purification that are less costly and highly efficient. In this work, we evaluated three different methods for lipase purification from Yarrowia lipolytica, such as precipitation using ammonium sulfate, ethanol, or acetone; aqueous two-phase systems (ATPS) based on polyethylene glycol (PEG) and potassium phosphate; and direct immobilization. It was impossible to obtain stable precipitates of the enzyme due to the low concentration of total protein and the presence of biosurfactant produced by the microorganism. Different mixture compositions were selected for the partitioning study. Three ATPS showed selective partitioning of the target enzymes, i.e., lipase and protease migrated to opposite phases. In the ATPS composed of 13 wt% PEG-4000 and 10 wt% salts, it was possible to achieve a purification factor for lipase of 4.2. Purification by immobilization performed by lipase-lipase interactions showed three lipases of distinct sizes in the crude extract. In the immobilization method by hydrophobic supports, phenyl-agarose and butyl‑agarose were more selective in immobilizing than octyl-agarose. In the ion exchange immobilization method, only the lipases identified at 66 kDa and 41 kDa have an attraction for DEAE-agarose (anionic) and sulfopropyl-agarose (cationic) matrices