Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vi, 84 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 76-83).

Th.--Pharm.--Paris 5, 1980. N°: 40.

Two forms of gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of two species of Thai catfish, Clarias inacrocephalus and C. batrachus. The peptides were detected using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA), The amino acid sequences of both forms were determined using Edman degradation. One form of GnRH in the brain-pituitary tissues of the Thai catfish was novel, whereas the second form of GnRH was identical to chicken GnRH-XI. The presence of the N-terminal pGlu residue in both peptides was established by digestion with pyroglutamyl aminopeptidase. In addition, catfish GnRH-I was studied by mass spectrometry. The localization of these two peptides was determined to be in the discrete brain areas and in the pituitary of female and male catfish, C. macrocaphalus, using heterologous and homologous radioimmunoassays. Initially a heterologous RIA was used with mammalian GnRH as iodinated tracer and standard, and an antiserum made against salmon GnRH. Catfish GnRH-I (novel form) was found in most areas of the female and male brain with the highest content and concentration in the female pituitary and in the male hypothalamus,, Catfish GnRH-II (chicken GnRH-II) was found with the highest content in the female Cerebellum and highest concentration in the pituitary, catfish GnRH-II (chicken GnRH-II) was found with the highest content and concentration for males in the same area, hypothalamus. Additionally, a homologous RIA was used with catfish GnRH-II (chicken GnRH-II) as iodinated tracer and standard, and an antiserum prepared against chicken GnRH-II. Catfish GnRH-II was detected with the highest content and concentration in the cerebellum of both sexes. These values are higher than the results obtained in the heterologous assay. The location of catfish GnRH-I suggests that it plays a role in regulating the release of gonadotropin from the pituitary since the high content and concentration of this immunoreactive GnRH are detected in the hypothalamus and pituitary gland. In contrast, catfish GnRH-II may act as a neurotransmitter in the catfish brain, in particular in tha cerebellum where a high content and concentration of immunoreactive GnRH are detected. Physiological in vivo studies indicate that catfish GnRH-II is more effective than catfish GnRH-I and other forms of GnRH such as mammalian and dogfish GnRH for induction of ovulation in catfish, C. macrocephalus. Eight GnRH analogs had varying potencies for the induction of ovulation, but the most effective forms were two forms of catfish GnRH-II (chicken GnRH-II) modified in positions six and ten. In vitro studies found that catfish GnRH-I not only causes the release of gonadotropin but also the release of growth hormone in a dose-dependent manner. The primary structures of the two catfish GnRH peptides are important for understanding the evolution of this family peptide. The novel catfish GnRH shows that only positions 5, 7 and 8 vary in the GnRH molecule in jawed vertebrates, whereas catfish GnRH-II provides direct evidence that the structure of this GnRH is conserved in teleosts. / Graduate

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Marine mussels accumulate amino acids and glucose from seawater against considerable concentration gradients. The principal site for this uptake is the gill. Previous studies using intact, isolated gills from marine mussels have suggested that the transport mechanism involves coupling to Na⁺, similar to the mechanism of secondary active transport of amino acids and glucose in vertebrate epithelia, but until this dissertation there had been no rigorous test of this hypothesis. Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel, Mytilus edulis, by differential and sucrose density centrifugation. The preparation procedure isolated a population of membranes enriched in brush border membrane markers. The transport of amino acids by two pathways, the alanine-lysine pathway (AK) and the alanine-proline pathway (AP), and the uptake of glucose was studied in the BBMV. The mechanism of transport through the three transport pathways is BBMV involved coupling to Na⁺. Concentrative uptake through the AK pathway, which transported alanine and lysine, also occurred in the presence of Li⁺ and K⁺ gradients. This pathway was the major route for alanine transport in BBMV. The AP pathway transported alanine and proline, and was strictly dependent on Na⁺. Glucose transport in gill BBMV resembled quite closely the Na⁺-coupled transport of glucose in vertebrate epithelia in such characteristics as Na⁺ and substrate specifically, and electrogenicity. Transport through the two amino acid uptake pathways (AK and AP) and through the glucose uptake pathway could be described by Michaelis-Menten kinetics, with high substrate affinities (K(t)'s below 10 μM). Furthermore, it is likely that multiple Na⁺ ions are involved in the transport of these amino acids and glucose in mussel gill BBMV. It appears that these transporters are adapted for function at low substrate concentrations and against large concentration gradients.