Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 125-162). Also available on the Internet.

Myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis was the only previously identified myokinin receptor. A cDNA encoding a neuropeptide receptor was cloned from the southern cattle tick, Boophilus microplus. The deduced amino acid sequence was 40 % identical to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative RT-PCR. When expressed in mammalian CHO-K1 cells, myokinins at nanomolar concentrations induced increases in intracellular calcium as measured by fluorescent cytometry. The rank order of potency for peptides tested was FFFSWS-NH2≥FFFSWG-NH2≥FFSWG-NH2>FYSWG-NH2>muscakinin>lymnokinin>>APTGFFGVR-NH2. The receptor coupled to a pertussis toxin insensitive G protein. Absence of extracellular calcium did not inhibit the calcium response, indicating the release of Ca2+ from intracellular stores. Receptor transcript was detected by RT-PCR in the dissected synganglia, ovaries, salivary glands, guts and Malpighian tubules of partially engorged adult female ticks. It is concluded that the B. microplus receptor is the first myokinin receptor cloned from an arthropod, and the first neuropeptide receptor known from the Acari. The presence of this receptor transcript in multiple tissues and all life stages suggests a multifunctional role in ticks.

The regulation of the immune system is complex, with many factors involved in controlling immune cell development, activation and homeostasis. These factors include neuropeptides as well as classic immunoregulatory molecules such as cytokines, chemokines and hormones. Neuropeptides and tachykinins in particular are known to be involved in immune response modulation through a cascade of events including vasodilation, plasma extravasation, the activation of immune cells, the secretion of pro-inflammatory cytokines and the recruitment of more immune cells. Furthermore, there is growing evidence that tachykinins play a role in hematopoiesis with Substance P as the proposed effector molecule. In 2000, our lab discovered a new tachykinin with remarkable structural similarity to SP and SP-like neurokinin receptor binding affinity. This molecule was designated Hemokinin-1 due to its expression in hematopoietic cells and its function in B cell development. Further gene expression analysis of HK-1 reveals a wide expression pattern although HK-1 transcripts are found predominantly in peripheral tissues while SP is mainly expressed in neuronal tissue. Based on this differential expression pattern, it has been suggested that HK-1 may act as the peripheral tachykinin and may have functions distinct from SP. In addition, given the crossreactivity of the SP antibodies to HK-1, it is important to determine whether HK-1 is the actual mediator of some functions previously attributed to SP. In this thesis, we examine the differential expression pattern of HK-1 to determine molecular mechanisms of regulation of HK-1 transcription and ultimately provide clues to its function in the immune system. In our analysis of the HK-1 promoter, we found a major difference in the basic transcriptional control of HK-1 and SP at the level of transcription initiation and identified several transcription factors including CREB and NFκB involved in regulating TAC4 gene expression in immune cells. Data presented in this thesis also reveal that the HK-1 gene is a direct target of Early B-cell Factor, a transcription factor known to activate B cell-specific genes as well as genes involved in adipogenesis and neuronal development. Our results show EBF regulates HK-1 gene expression in differentiating B cells as well as a monocytic cell line. Our data indicate EBF may also be responsible for the high levels of HK-1 transcript in the olfactory epithelium, suggesting a bridge between the nervous system and the immune system.

Zugl.: Giessen, Univ., Diss., 2005

Universiẗat, Diss., 2005--Giessen.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

In Drosophila, eight insulin-like peptides (DILP1-8) are produced and secreted in different locations. They regulate many aspects of development and physiology, such as organism growth, metabolic homeostasis, reproduction, stress resistance and life span. DILP2, 3 and 5 are mainly produced by a cluster of median neurosecretory cells in the brain known as insulin producing cells (IPCs). Here we showed that IPCs are under tight regulation of two G-protein coupled receptors (GPCRs), serotonin receptor 5-HT1A and octopamine receptor OAMB. Genetic manipulations of these two receptors in IPCs affected transcription levels of DILPs, hence altered feeding, carbohydrate levels, and resistance to stress (Paper I and II). Moreover, we showed that the insulin receptor (dInR) is strongly expressed in leucokininergic neurons (LK neurons), and selectively regulates growth of around 300 neuropeptidergic neurons expressing the bHLH transcription factor DIMMED. Overexpression of dInR in DIMM-positive neurons led to substantial neuronal growth, including cell body size, golgi apparatus and nuclear size, while knockdown of dInR had the opposite effect (Paper III). Manipulations of components in the insulin signaling pathway in LK neurons resulted in the similar cell size phenotypes. Furthermore, dInR regulated size scaling of DIMM-postive neurons is nutrient-dependent and partially requires the presence of DIMM (Paper III). Finally, we investigated the roles of DILPs (2, 3, 5 and 7) and LK neurons in regulation of feeding and diuresis at the adult stage (Paper IV).  In summary, we have identified two more regulators for IPC activity and demonstrated developmental roles of  DILPs and dInR in regulating neuronal size. Moreover, DILPs regulate water homeostasis together with a diuretic hormone leucokinin and as a consequence affects feeding behavior. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press. Paper 4: Manuscript.</p>

α-MSH immunoreactive peptides were fractionated and characterized in rat and human brain and rat pituitary by reversed phase high pressure liquid chromatographic techniques. α-MSH and deacetylated α-MSH were two major naturally existing peptides in both brain and pituitary gland. Subsequent experiments examined the roles of these two peptides in neuronal function. The α-MSH was clearly more effective than deacetylated α-MSH in improving performance on a visual discrimination task after intraperitoneal administration and in inducing excessive grooming after intraventricular administration. The difference in behavioral potency may be explained by the fact that α-MSH was much more resistant to peptidase degradation than was deacetylated α-MSH. N-acetylation of α-MSH may be an effective regulatory process for modulating the behavioral potency of the secretory product of α-MSH-containing pituitary cells and neurons.

Electrophysiological studies of myenteric neurons in the guinea-pig antrum suggest that different neuroactive compounds are involved in synaptic transmission. It is not known what neurotransmitters and neuropeptides are present and to what extent they colocalize. Immunohistochemical stainings were performed on whole-mount preparations of the guinea-pig antrum. Immunoreactivity for neuron-specific enolase was used as a general marker and was set at 100%. There was no overlap between cholinergic and nitrergic neurons, resulting in two separate subpopulations. The presence of choline acetyltransferase immunoreactivity was used to identify the cholinergic subset, which accounted for 56% of the cells. Immunoreactivity for nitric oxide synthase, on the other hand, was displayed in 40.7% of the neurons. Substance-P immunoreactivity was present in 37.4% of the cells and vasoactive intestinal peptide and neuropeptide Y in 21.7% and 28.6%, respectively. Small subsets of neurons had immunoreactivity for serotonin (3.9%), calretinin (6.8%) and calbindin (0.5%). Colocalization studies revealed several subgroups of neurons, containing one or more of the screened markers. Though some similarity is found in the chemical coding of the antrum compared to that of the small intestine and the corpus, remarkable differences can be seen in the occurrence of some subpopulations. Cholinergic neurons are not as predominant as in other parts of the gut, serotonin presence is doubled and some vasointestinal-peptide-positive neurons express substance P. These differences might reflect the highly specialized function of the antrum; however, the exact role of these classes remains to be established.