Neurotensin (NT) is a brain-gut peptide, localised to mucosal endocrine N-cells in the gastrointestinal tract. The most potent stimuli for NT release are ingested fats. NT, in vivo, inhibits the migrating myoelectric complex and stimulates colonic motility. The aim of this thesis was to investigate NT and its receptors in human isolated intestine, focusing on motility aspects. ???Normal??? colon and ileum were obtained from patients undergoing resection for carcinoma, and ???disease??? colon was obtained from patients undergoing resection for ulcerative colitis (UC), Crohn???s disease (CD), diverticular disease (DD) and slow transit constipation (STC). In functional experiments with ???normal??? ascending and sigmoid colon, NT contracted taenia coli strips by direct mechanisms (EC50 ??? 100 nM). In circular muscle (CM) strips, contractions were mediated by NT-induced release of enteric mediators (indirect actions), which were regionally specific, as well as by direct actions on the smooth muscle. In contrast, in ???normal??? terminal ileum, the predominant action of NT was a potent (EC50 ??? 3 nM), directly mediated inhibition of spontaneous contractions in longitudinal muscle strips. NT receptors were characterised using radioligand binding with [125I]-NT and [3H]-NT, and localised using autoradiography and immunohistochemistry and confocal microscopy. In the sigmoid colon, binding sites corresponded to NTS1 receptors and could be visualised on the smooth muscle and the enteric ganglia, supporting both direct and neuronal actions for NT, respectively. In the ileum, a similar distribution of binding sites could be visualised. In gastrointestinal disease, NT contractile responses were reduced in UC, DD and STC colon CM strips, but were unchanged in CD. In contrast, NT responses in all diseases were largely unchanged in TC strips, suggesting disease/treatment-induced alterations in NT mechanisms. In UC, cholinergic mechanisms appeared to be altered, whereas in DD disease, prostaglandin mechanisms were affected. In STC, there was an apparent loss of a normally predominant component in NT-mechanisms, but contractile mechanisms appeared to be retained. In summary, these studies demonstrated regional and muscle layer specific actions and mechanisms of action for NT, and support alterations in enteric signaling in disease.

Neurotensin (NT) is a brain-gut peptide, localised to mucosal endocrine N-cells in the gastrointestinal tract. The most potent stimuli for NT release are ingested fats. NT, in vivo, inhibits the migrating myoelectric complex and stimulates colonic motility. The aim of this thesis was to investigate NT and its receptors in human isolated intestine, focusing on motility aspects. ???Normal??? colon and ileum were obtained from patients undergoing resection for carcinoma, and ???disease??? colon was obtained from patients undergoing resection for ulcerative colitis (UC), Crohn???s disease (CD), diverticular disease (DD) and slow transit constipation (STC). In functional experiments with ???normal??? ascending and sigmoid colon, NT contracted taenia coli strips by direct mechanisms (EC50 ??? 100 nM). In circular muscle (CM) strips, contractions were mediated by NT-induced release of enteric mediators (indirect actions), which were regionally specific, as well as by direct actions on the smooth muscle. In contrast, in ???normal??? terminal ileum, the predominant action of NT was a potent (EC50 ??? 3 nM), directly mediated inhibition of spontaneous contractions in longitudinal muscle strips. NT receptors were characterised using radioligand binding with [125I]-NT and [3H]-NT, and localised using autoradiography and immunohistochemistry and confocal microscopy. In the sigmoid colon, binding sites corresponded to NTS1 receptors and could be visualised on the smooth muscle and the enteric ganglia, supporting both direct and neuronal actions for NT, respectively. In the ileum, a similar distribution of binding sites could be visualised. In gastrointestinal disease, NT contractile responses were reduced in UC, DD and STC colon CM strips, but were unchanged in CD. In contrast, NT responses in all diseases were largely unchanged in TC strips, suggesting disease/treatment-induced alterations in NT mechanisms. In UC, cholinergic mechanisms appeared to be altered, whereas in DD disease, prostaglandin mechanisms were affected. In STC, there was an apparent loss of a normally predominant component in NT-mechanisms, but contractile mechanisms appeared to be retained. In summary, these studies demonstrated regional and muscle layer specific actions and mechanisms of action for NT, and support alterations in enteric signaling in disease.

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 158 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 122-155).

No description available.

Neurotensin (NTS) is a tridecapeptide which is widely distributed in the central nervous system and digestive tract. It is highly expressed in neurons of the hypothalamus, a region of the brain known to control feeding behavior. Several studies have shown that intracerebroventricular injection of NTS decreased food intake in rats. NTS was therefore characterized and analyzed for associations with growth and carcass traits in beef cattle.<p>NTS mRNA was successfully isolated from brain, spinal cord, abomasum, rumen wall, small intestine and skin samples. The complete bovine mRNA sequence was obtained from skin, along with partial genomic sequence. Three single nucleotide polymorphisms (SNPs) in the 3 untranslated region (3UTR) and six intronic SNPs were identified. The three SNPs in the 3UTR were not in linkage disequilibrium. Of the three SNPs in the 3UTR, two had minor allele frequencies of 2% and therefore were not analyzed further. The minor allele frequencies for the third SNP (*419G>A) ranged between 0% and 23% for four major beef breeds. The *419G>A SNP was also used to map NTS to bovine chromosome five between markers BM6026 (13 cM, LOD=4.03) and RM103 (4 cM, LOD=3.63).<p>No significant associations between the *419G>A SNP and growth traits were identified. Statistical analysis revealed significant genotype associations for rib eye area (REA), grade fat and moisture in the Canadian Beef Reference Herd (CBRH). These associations were not verified in a second group of purebred yearling bulls. However, significant associations with end of trial fat, %fat and fat deviation were associations for marbling and quality grade. In all cases the AA genotype was associated with increased fat.<p>Although significant associations between carcass measurements and genotype at the *419G>A SNP were present in some populations, none of these associations were found in more than one population. It was therefore concluded that the *419G>A SNP on the bovine NTS gene does not prove to have an economic advantage to the beef cattle industry.

Neurotensin (NTS) is a tridecapeptide which is widely distributed in the central nervous system and digestive tract. It is highly expressed in neurons of the hypothalamus, a region of the brain known to control feeding behavior. Several studies have shown that intracerebroventricular injection of NTS decreased food intake in rats. NTS was therefore characterized and analyzed for associations with growth and carcass traits in beef cattle.<p>NTS mRNA was successfully isolated from brain, spinal cord, abomasum, rumen wall, small intestine and skin samples. The complete bovine mRNA sequence was obtained from skin, along with partial genomic sequence. Three single nucleotide polymorphisms (SNPs) in the 3 untranslated region (3UTR) and six intronic SNPs were identified. The three SNPs in the 3UTR were not in linkage disequilibrium. Of the three SNPs in the 3UTR, two had minor allele frequencies of 2% and therefore were not analyzed further. The minor allele frequencies for the third SNP (*419G>A) ranged between 0% and 23% for four major beef breeds. The *419G>A SNP was also used to map NTS to bovine chromosome five between markers BM6026 (13 cM, LOD=4.03) and RM103 (4 cM, LOD=3.63).<p>No significant associations between the *419G>A SNP and growth traits were identified. Statistical analysis revealed significant genotype associations for rib eye area (REA), grade fat and moisture in the Canadian Beef Reference Herd (CBRH). These associations were not verified in a second group of purebred yearling bulls. However, significant associations with end of trial fat, %fat and fat deviation were associations for marbling and quality grade. In all cases the AA genotype was associated with increased fat.<p>Although significant associations between carcass measurements and genotype at the *419G>A SNP were present in some populations, none of these associations were found in more than one population. It was therefore concluded that the *419G>A SNP on the bovine NTS gene does not prove to have an economic advantage to the beef cattle industry.

Thesis (Ph.D.)--Tufts University, 2004. / Adviser: David E. Cochrane. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 137-165). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are zincdependent metallopeptidases that metabolize small bioactive peptides. The two enzymes share60 % sequence identity and their crystal structures demonstrate that they adopt nearly identicalfolds. They generally cleave at the same sites, but they recognize different positions on somepeptides, including neurotensin, a 13-residue peptide involved in modulation of dopaminergiccircuits, pain perception, and thermoregulation.On the basis of crystal structures and previous mapping studies, four residues(E469/R470, M490/R491, H495/N496, and R498/T499, TOP residues listed first) in thesubstrate-binding channel appear positioned to account for differences in specificity. TOPmutated to the neurolysin residues at all four position cleaves neurotensin at the neurolysin siteand neurolysin mutated to the TOP residues at all four sites cleaves at the TOP position. Using aseries of constructs mutated at only three sites, it was determined that only two of the mutations,E469/R470 and R498/T499, are required to swap the specificity of TOP and neurolysin. Theseresults were confirmed by testing the two mutation constructs, and either single mutant of TOPshown an intermediate specificity, cleaving at both sites.Crystal structures of the two mutation constructs of TOP and neurolysin unligandedforms, the mutations do not perturb local structure, but side chain conformations at theR498/T499 position differ from those of the mimicked enzyme. A model for differentialrecognition of neurotensin based on differences in surface charge distribution in the substratebinding sites is proposed. The model is supported by finding that reducing the positive charge onthe peptide results in cleavage at both hydrolysis sites.This dissertation also includes a description of the production and crystallization trials ofhuman neprilysin (E.C. 3.4.24.11), which will be used as another model system for studyingspecificity in metallopeptidases. In addition, the production and crystallization, and crystalcharacterization of human choline acetyltransferase (EC 2.3.1.6) is described.

Η Νευροτενσίνη (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) [NT] είναι ένα πεπτίδιο δεκατριών αμινοξέων, το οποίο απομονώθηκε για πρώτη φορά από τον υποθάλαμο βοοειδούς και αργότερα από τα έντερα. Η ΝΤ έχει ευρύ φάσμα βιολογικών δράσεων τόσο στο κεντρικό όσο και στο περιφερικό νευρικό σύστημα. Από τις δράσεις αυτές συμπεραίνεται ο διπλός ρόλος της ως νευροδιαβιβαστή/νευρορυθμιστή στον εγκέφαλο και ως ορμόνη/κυτταρικός μεσολαβητής σε περιφερικούς ιστούς. Οι φυσιολογικές και βιοχημικές δράσεις της ΝΤ εκδηλώνονται μέσω της αλληλεπίδρασής της με τους υποδοχείς της. Μέχρι σήμερα τρεις υπότυποι του υποδοχέα (NTS1, NTS2 & NTS3) έχουν κλωνοποιηθεί. Και οι τρεις υπότυποι αναγνωρίζουν και δεσμεύουν το C-τελικό εξαπεπτίδιο της Νευροτενσίνης [NT(8-13)], το οποίο αποτελεί το μικρότερο τμήμα της πεπτιδικής αλληλουχίας με πλήρη βιολογική δράση. Η γρήγορη όμως αποικοδόμηση της NT(8-13) από την δράση των πρωτεολυτικών ενζύμων οδηγεί στην αναγκαιότητα σύνθεσης αναλόγων τα οποία θα διατηρούν υψηλή ικανότητα σύνδεσης με τους υποδοχείς και παράλληλα να παρουσιάζουν ενζυμική σταθερότητα. Στην παρούσα μελέτη παρουσιάζεται η σύνθεση νέων αναλόγων της NT(8-13) με τη χρήση της Fmoc/But μεθοδολογίας επί στερεάς φάσεως χρησιμοποιώντας ως στερεό υπόστρωμα την 2-χλωροτριτυλο ρητίνη. Επίσης, πραγματοποιήθηκαν πειράματα δέσμευσης σε HT-29 κυτταρική σειρά, η οποία εκφράζει ενδογενώς τους υποδοχείς της ΝΤ. Τέλος πραγματοποιήθηκε η σύγκριση των διαμορφώσεων δύο κυκλικών αναλόγων με την χρήση της φασματοσκοπίας NMR και Μοριακής Δυναμικής. / Neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) [NT] is a tridecapeptide originally isolated from bovine hypothalamus and later from intestines. NT displays a wide spectrum of biological actions both in the central and peripheral nervous systems of different mammalian species. These actions have led to the proposal that this peptide fulfills a dual function as a neurotransmitter/neuromodulator in the brain and as a hormone/cellular mediator in peripheral tissues. The physiological and biochemical actions of NT are mediated through binding to NT receptors (NTRs). Up to now, three subtypes of neurotensin receptors (NTS1, NTS2 & NTS3) have been cloned. All three receptors recognize the same C-terminal hexapeptide fragment of NT [NT(8-13)], which corresponds to the shorter fragment of NT that maintains full biological activities. Although NT(8-13) possesses high receptor binding affinity, it is rapidly degraded by peptidase action. Therefore, it is important to synthesize analogues with stabilized bonds against metabolic deactivation which do not lose binding affinity. Based on these findings, we herein report the synthesis of new analogues of NT(8-13) with modifications in the basic structure needed for high affinity binding in order to improve the metabolic stability. The analogues were synthesized by the Fmoc/But solid phase methodology utilizing 2-chlorotrityl chloride resin. Electrospray MS was in agreement with the expected results. Competition radioligand binding studies were performed in membrane homogenates from HT-29 cells endogenously expressing NTRs. A series of NMR spectra including 1H, 2D TOCSY and 2D NOESY were recorded on a Varian 600MHz spectrometer for two cyclic analogues in order to elucidate their structure. The conformation properties of the two peptides, as well as similarities and differences between them are currently studied using NMR spectroscopy and Molecular Dynamics simulations.

No description available.