No description available.

No description available.

No description available.

It is well known that information contained in a protein sequence is what allows it to fold into its three-dimensional shape, which performs a specific function. It has been possible for some time to search for proteins with similar sequences, using bioinformatics tools such as BLAST. But it is also known that proteins with similar, or even the same sequence can adopt different structures and vice-versa. With this in mind, we look to use a method called Rosetta-HMMER to perform conformationally specific sequence searches in order to exploit this property of proteins. This method involves the use of Rosetta to redesign protein structures to fit a specified α-carbon backbone, and then uses HMMER to generate a sequence profile. This profile can then be used to query for sequences able to adopt the specified backbone structure. These collected sequences can then be aligned for the purpose of performing statistical coupling analysis. We have used this Rosetta-HMMER method in conjunction with available structures of the α7 acetylcholine receptor to show that distinct sequence profiles generated from different conformations of the same protein are capable of retrieving unique sets of natural sequences when used as a query. We have also shown that when these unique sets of natural sequences are used to perform statistical coupling analysis, different residues are identified as statistically coupled, potentially generating insight into residues that have more potential importance for one backbone conformation over another.

The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.

No description available.

Prototoxin proteins have been identified as members of the Ly6/uPAR super family whose three-finger motif resembles that of α-bungarotoxin. Though they are known to modify the function of nAChRs, their specificity is still unclear. Our lab identified three prototoxin proteins in the chicken ciliary ganglion: Ch3ly, Ch5ly, and Ch6ly. Ch6ly was later identified as prostate stem cell antigen (PSCA), and specifically decreased the amount of calcium influx through the homomeric α7 nAChR subtype. I then identifiedCh3ly and Ch5ly as LY6E and LYPD6B, respectively. I focused my attention onLYPD6B because of its expression in the brain. This dissertation tests whether LYPD6Bis a prototoxin protein that specifically co-localizes with and modifies the function of the heteromeric α3β4* nAChRs (the other nAChR subtype expressed in the chicken ciliary ganglia). In the first part of my dissertation I performed intracellular two-electrode voltage clamp on Xenopus oocytes co-expressing human LYPD6B and different stoichiometries of the α3β4* nAChR, these included two (α3)2(β4)3 withβ4−α3−β4−β4−α3 and β4−α3−β4−α3−β4 stoichiometries, two (α3)3(β4)2 with stoichiometries β4−α3−α3−β4−α3 and β4−α3−β4−α3−α3, two (α3β4)2(α5D)β4−α3−α5D−β4−α3 and β4−α3−β4−α3−α5D, and (α3β4)2(α5N) with stoichiometries β4−α3−α5N−β4−α3 and β4−α3−β4−α3−α5N. Concatemeric constructs are designed to link nAChR subunits, thus when translated it is done so as a single polypeptide. LYPD6Bincreased the acetylcholine (ACh) potency and desensitization rate, but decreased the maximum current response (Imax) for the (α3)3(β4)2 nAChR subtype. Yet, LYPD6Bonly decreased the Imax for the (α3β4)2α5 D-variant and not the N-variant (associated with increase nicotine consumption). For the second part of my dissertation, I determined if the expression of LYPD6B correlated with nAChRs in an activity dependent manner. Though LYPD6B mRNA expression correlates with nAChR subunit mRNA expression levels, it seemed to be independent of nAChR activity. To determine if fluorescent colocalization occurs between LYPD6B and a specific nAChR subtype, I genetically engineered LYPD6B to express a human influenza hemagglutinin (HA) epitope tag and cloned into a chicken retrovirus. LYPD6B was shown to co-localize only with the α3β4*heteromeric and not the homomeric α7 nAChRs, in a nAChR activity dependent manner. This study adds to the complexity of a prototoxin’s function by suggesting that the specificity is dependent on nAChR type and stoichiometry. It is the first in identifying a prototoxin protein, LYPD6B, which specifically modulates the function of the(α3)3(β4)2 and (α3β4)2(α5 D-variant) heteromeric nAChR subtypes. For the (α3β4)2(α5D-variant) nAChR subtype LYPD6B decreased the Imax. Such observation may be telling of a novel mechanism involved with nicotine dependence. For the(α3)3(β4)2 nAChR subtype LYPD6B increases its ACh sensitivity, desensitization rate, while decreasing Imax. Additionally, the co-localization of LYPD6B and α3β4* nAChRsin the lack of nAChR activity highlights the relevance of the functional effects α3β4*nAChRs exhibit due to LYPD6B. Such relevance may be the utilization of limiting Ach amounts.

Pontomesencephalic tegmental cholinergic neurons were destroyed in cats by local injections of kainic acid in order to assess the role of these neurons in sleep-wakefulness states and in the defining variables of these states: EEG (electroencephalographic) and EMG (electromyographic) amplitude, PGO (ponto-geniculo-occipital) spike rate, REMs (rapid eye movements) and (OBS) olfactory bulb spindles. Loss of cholinergic innervation to forebrain and brainstem structures was also assessed by histochemistry. Histological and histochemical analysis of the brains after the lesion showed a major destruction of the pontomesencephalic cholinergic neurons and a major loss of innervation to thalamic nuclei and brainstem regions, including the reticular formation. Whereas the states of waking and slow wave sleep were relatively unaffected, paradoxical sleep (PS) was reduced or eliminated immediately following the lesions. Two to three weeks later, incipient PS-like episodes returned with a reduced PGO spike rate and REMs, and an elevated EMG amplitude, marking the loss of muscle atonia. Such results suggest pontomesencephalic cholinergic neurons and their projections to thalamic and brainstem regions are important for the expression of PS and its defining variables.

Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Nael A. McCarty; Committee Co-Chair: Dr. Darrell Jackson; Committee Member: Dr. Alfred H. Merrill; Committee Member: Dr. Barbara D. Boyan; Committee Member: Dr. Harish Radhakrishna; Committee Member: Dr. Marion B. Sewer

No description available.