Showing 1 - 2 of 2 Items
Characterization of O-Linked Glycosylated Neuropeptides in the American Lobster (Homarus americanus): The Use of Peptide Labeling Following Beta Elimination
Date: 2020-01-01
Creator: Edward Myron Bull
Access: Open access
- Neuropeptides are a class of small peptides that govern various neurological functions, and the American lobster (Homarus americanus) provides a model system for their characterization. Neuropeptides are commonly post-translationally modified (PTM), and one common PTM is glycosylation. Past research in the Stemmler lab has found glycosylated neuropeptides in H. americanus; however, the extent and biological role of this modification has not been well characterized. This study was undertaken to determine the number of glycosylated peptides in the sinus glands of H. americanus and to develop an approach to tag the site of glycosylation using beta-elimination chemistry. LC-MS paired with high pH reverse phase fractionation was used to survey for glycosylated neuropeptides and beta elimination with an amine tag was used as an approach to characterize the site of glycosylation. Our results indicate that high pH fractionation is a useful approach to simplify complex mixtures of neuropeptides and improve glycopeptide detection. Efforts to use beta elimination and tagging to characterize glycosylated neuropeptides have been less successful. Beta elimination of full length peptides resulted in peptide degradation. An approach utilizing chymotrypsin to reduce peptide size coupled with beta elimination and labeling with 2-dimethylaminoethanethiol showed less evidence for degradation, and this approach yielded data isolating two potential serine residues for the site of glycosylation; however, the data was not sufficient to distinguish the two sites. Work to optimize reaction conditions using a glycopeptide standard showed that multiple isomeric products were formed during beta elimination. With the goal of optimizing reaction conditions, future work will further examine reaction kinetics to eventually apply the approach to the entire sinus gland
Rhythmic behaviors: Understanding neuromodulation at the neuromuscular level
Date: 2023-01-01
Creator: Kenneth Garcia
Access: Open access
- Neuromodulation allows for the flexibility of neural circuit dynamics and the outputs they produce. Studies of the stomatogastric nervous system (STNS) have expanded our knowledge on the actions of neuromodulators, small molecules that most often activate G-protein coupled receptors and reconfigure circuit activity and composition. In these systems, modulation has been found to occur at every level, from sensory-motor coupling to neuromuscular transmission (Harris-Warrick and Marder 1991). Neuromodulators have complex effects on motor output; they can alter the firing of individual neurons while also modulating muscle properties, neuromuscular transmission, and sensory neuron response to muscle activity (Fort et al. 2004). We investigated this further by recording the motor output produced by the gastric mill rhythm of the lobster STNS under neuromodulator conditions. How is this neuromuscular system as a whole modulated to produce motor flexibility? We hypothesized that these neuromodulators act on individual receptors of component neurons of central pattern generator (CPG)-effector system themselves and at the periphery, coordinately altering muscle contraction by altering all levels of the crustacean neuromuscular system. Application of NRNFLRFamide, RPCH, oxotremorine, and proctolin to the gastric mill 4 (gm4) muscles of the Cancer crab showed that neuromodulators that have been found to have variable, yet significant effects on the activity of the neurons of the STNS directly alter the activity of the gm4 muscles as well, suggesting that coordination of peripheral actions and direct neuronal modulation regulates patterned motor output.