Thesis (Ph. D.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 173-194).

Thesis--Auburn University, 2009. / Abstract. Vita. Includes bibliographical references (p. 52-75).

Cloudman S91 melanoma cells respond to a variety of endocrine factors, including melanotropins and steroid hormones. The murine S91 melanoma cell line, CCL 53.1, responded to α-melanocyte-stimulating hormone (α-MSH) in a dose- and a time-dependent manner, by increased tyrosinase activity. The minimal effective dose of α-MSH required to stimulate tyrosinase activity was 10⁻⁹M. By prolonging the exposure period of the cells to the hormone from 24 to 48, to 72 hours, the magnitude of tyrosinase stimulation was increased, but the minimal effective dose was not changed. α-MSH action involved elevation of intracellular cyclic AMP levels, and did not require the influx of extracellular calcium. Three melanotropin analogues, [Nle⁴, D-Phe⁷]-α-MSH, Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₁-NH₂, and Ac-[Nle⁴, D-Phe⁷]- α-MSH₄₋₁₀-NH₂, have been shown to be more active than (alpha)-MSH in dispersing melanosomes within amphibian and reptilian integumental melanophores. These analogues also demonstrate prolonged activities and resistance to enzymatic degradation. The unique properties of these melanotropin analogues led to investigate their effects on the phenotypic expression and on the proliferation of S91 melanoma cells. The relative potency and the possible prolonged actions of the three [Nle⁴, D-Phe⁷] -substituted analogues were investigated and compared to those of α-MSH. The melanotropin analogues proved to be 100-1000 fold more active than α-MSH in stimulating tyrosinase activity. These analogues elicited significant tyrosinase activation following brief contact times with the cells, and maintained their stimulatory effects for days after removal from the culture flasks, and after the α-MSH effect had totally dissipated. Contrary to previous reports that melanotropins inhibit the proliferation of melanoma cells, α-MSH and [Nle⁴, D-Phe⁷] -α-MSH stimulated, rather than inhibited, the proliferation of CCL cells under culture conditions that allowed optimal phenotypic expression. Also, the effects of the steroids, β-estradiol, progesterone, and dexamethasone, on CCL cells were studied. Dexamethasone had the most remarkable effects, which involved stimulation of tyrosinase activity and inhibition of proliferation in monolayer culture and in soft agar. Furthermore, this study defined some of the factors that influence the endocrine responsiveness of melanoma cells. The results of this study have important implications for the regulation of phenotypic expression and of proliferation in S91 melanoma cells, and for the properties of the melanoma melanotropin receptor.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Alpha-melanotropin (alpha-melanocyte stimulating hormone, α-MSH) exerts its biological action by binding to specific receptors on the outer cell membranes of its target tissues with a high degree of affinity and specificity. Current evidence suggests that this takes place both in vitro and in vivo in both normal and malignant melanocytes. Thus, if it were possible to attach a radioisotope (e.g., ¹²⁵I) to α-MSH, or a suitable analogue, without interfering with the receptor affinity of the hormone, then a radioreceptor assay could be developed which would allow hormone-receptor interaction to be studied in detail. In addition, this radio-labeled melanotropin might be expected to accumulate in melanoma tumors in vivo thus facilitating tumor localization by nuclear imaging methods as has been successfully accomplished for thyroid tumors. The present studies were initiated to develop a radioactive melanotropin with full, or nearly full, biological activity. This labeled melanotropin must be of sufficient specific radioactivity to be suitable as a tracer in a radioreceptor assay and ultimately as a marker for in vivo tumor localization. The studies described herein provide information concerning: chloramine T induced iodination, lactoperoxidase catelyzed iodination, and iodogen induced iodination of α-MSH and certain structural analogues. Radio-labeled derivatives of various melanotropins were prepared using a variety of iodination techniques. Under conditions commonly used for the iodination of other peptides a substantial loss of biological activity of the native hormone (α-MSH) was observed. This loss of hormonal activity was primarily a consequence of oxidation of methinonine and occurred regardless of the oxidant used (chloramine T, lactoperoxidase-hydrogen perioxide, or iodogen). Under similar iodination conditions using 4-norleucine-alpha-melanotropin ([Nle⁴]-α-MSH), satisfactory incorporation of label into the peptide was accomplished without significant loss of biological activity. Data are presented suggesting that this peptide is far superior to α-MSH for use in the preparation of a radioactive melanotropin. Although some success was achieved using [Nle⁴]-α-MSH with all three iodination methods, the simplest and most consistent method involved the use of iodogen followed by purification of the labeled product using high performance liquid chromatography (HPLC). This importance of these studies in the development of a tracer for a radio-receptor assay and for in vivo localization of melanoma tumors is discussed.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 153-156). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 153-156). Also available on the Internet.

Intermedin (IMD) is a peptide hormone discovered in 2004 belonging to the calcitonin/calcitonin gene-related peptide superfamily. It signals through a Gprotein coupled receptor by the coupling of a calcitonin receptor-like receptor (CRLR) and one of the receptor activity-modifying proteins (RAMPs) 1-3. Due to its similarity to adrenomedullin in structure and functions, IMD is also known as adrenomedullin 2 (ADM2). Among members of the superfamily, IMD shares the highest degree of homology with ADM, which is a multifunctional vasodilator ubiquitously expressed in various tissues and organs and has been studied by our group for its reproductive functions. It is hypothesized that IMD may be present in the reproductive systems of the rat and exert some effects on reproductive functions. The objectives of this study were to investigate the expression of IMD and its receptor components in the male and female reproductive systems of the rat, the changes in expression across the oestrous cycle, and its effect on uterine contraction. The gene expression levels of Imd and its receptor components and peptide levels of IMD were measured by RT-PCR and enzyme immunoassay respectively. The effect of IMD on the uterine contraction was studied by the organ bath technique. Imd mRNA and IMD levels were detected in the testis, epididymis, ventral prostate, coagulating gland, and seminal vesicle of the male rat and the ovary, oviduct, and uterus of the female rat, suggesting possible roles for IMD in both the male and female reproductive systems. In the male, the Imd mRNA levels were the highest in the seminal vesicle but lowest in the testis and the epididymis and IMD peptide levels were the highest in the coagulating gland but lowest in the epididymis. In the female, the Imd mRNA and IMD peptide levels were the highest in the oviduct and the uterus respectively while both the Imd and IMD levels were the lowest in the ovary. Imd mRNA and IMD levels displayed cyclic changes in various female reproductive tissues across the oestrous cycle. In the ovary, positive immunostaining was detected in the follicles and corpora lutea with more staining in the latter. The Imd mRNA level was significantly higher at prooestrus than dioestrus while the IMD peptide level was significantly higher at metoestrus than dioestrus. In the oviduct, the Imd mRNA level was the lowest at dioestrus but the IMD peptide level was the highest at dioestrus. Positive immunostaining was observed in the ciliated epithelial cells. Uterine Imd mRNA level was the highest at prooestrus while the IMD level was the highest at dioestrus. IMD was found in the luminal and glandular epithelia. IMD significantly reduced the uterine contraction amplitude and frequency but not the basal tone. CGRP receptor antagonist hCGRP8-37 and ADM receptor antagonist hADM22-52 partially abolished the inhibitory effect of IMD on uterine contraction while the IMD specific receptor antagonist hIMD17-47 completely blocked the actions. Enzyme inhibitors of NO (L-NAME) and PI3K (Wortmannin) pathways diminished the IMD-mediated effects on uterine contraction while cAMP/PKA blocker KT5720 had no effect. / published_or_final_version / Physiology / Master / Master of Philosophy

α-MSH (α-melanotropin) is a naturally occurring linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) that is primarily known for its ability to stimulate integumental melanocytes and more recently has been implicated in a variety of physiological and neurological processes. It has been shown that substitution of D-phenylalanine in the seven position of this hormone led to an analogue with increased potency and prolonged biological activity. Furthermore, cyclization between the four and ten positions via a cystine bridge led to analogues with enhanced potency. In this regard, a series of conformationally restricted linear and cyclic fragment analogues of α-MSH have been prepared and carefully analyzed by both biological and biophysical methods. Conformational restriction was incorporated in α-MSH fragment analogues, by: (1) substitution of sterically restricted amino acids into the native sequence; or (2) cyclization of the peptide via a disulfide bridge. Due to the biological differences observed for these synthetic α-MSH fragment analogues, a complete conformational analysis by both proton and carbon-13 NMR was performed. The conformational preferences of the backbone were examined by analyzing: (1) the alpha proton chemical shifts; (2) the amide proton chemical shifts; (3) the amide proton coupling constants; and (4) the amide proton temperature dependencies. The data suggests that the peptide backbone in both linear and cyclic analogues possesses a great amount of conformational flexibility with no hydrogen-bonded stabilization. The three-dimensional orientations of individual amino acid side chains have been examined by analyzing: (1) the chemical shifts of the side chain protons; (2) the alpha-beta coupling constants (corresponding rotamer populations); and (3) the carbon-13 spin lattice relaxation times (T₁). A careful examination a the chemical shifts of the side chains of individual amino acids in linear α-MSH fragments reveals that incorporation of an aromatic D-amino acid in the seven position results in an interaction of the side chains of the six, seven and eight positions. In addition, the low carbon-13 spin-lattice relaxation times for the β-carbons of the 5-9 sequence for both Ac-[Nle⁴]-α-MSH₄₋₁₁-NH₂ and Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₁-NH₂, provides further evidence for an interaction of these side chains. Similar shielding patterns have been observed for the cyclic α-MSH fragment analogues depending upon whether L- or D-phenylalanine is incorporated in the seven position. Considering the differences in biological potency and the similarities in the NMR parameters between the linear and cyclic homologs, it can be concluded that the conformational properties that determine biological potency are too subtle to be measured by present NMR methodology. Furthermore, the similarity of the NMR shielding patterns suggests that a 23-membered ring is too large to impart significant conformational constraints on the peptide backbone or amino acid side chains.

No description available.