Conserved molecular switch interactions in modeled cardioactive RF-NH2 peptide receptors: Ligand binding and activation.

Peptides may act through G protein-coupled receptors to influence cardiovascular performance; thus, delineating mechanisms involved in signaling is a molecular-based strategy to influence health. Molecular switches, often represented by conserved motifs, maintain a receptor in an inactive state. However, once the switch is broken, the transmembrane regions move and activation occurs. The molecular switches of Drosophila melanogaster myosuppressin (MS) receptors were previously identified to include a unique ionic lock and novel 3-6 lock, as well as transmission and tyrosine toggle switches. In addition to MS, cardioactive ligands structurally related by a C-terminal RF-NH2 include sulfakinin, neuropeptide F (NPF), short NPF, and FMRF-NH2-containing peptide subfamilies. We hypothesized receptor molecular switch motifs were conserved within a RF-NH2 subfamily and across species. Thus, we investigated RF-NH2 receptor (RFa-R) molecular switches in D. melanogaster, Tribolium castaneum, Anopheles gambiae, Rhodnius prolixus, and Bombyx mori. Adipokinetic hormone (AKH), which does not contain a RF-NH2, was also examined. The tyrosine toggle switch and ionic lock showed a higher degree of conservation within a RF-NH2 subfamily than the transmission switch and 3-7 lock. AKH receptor motifs were not representative of a RF-NH2 subfamily. The motifs and interactions of switches in the RFa-Rs were consistent with receptor activation and ligand-specific binding.

Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus.

We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents.

Long-term effects of a single exposure to immobilization on the hypothalamic-pituitary-adrenal axis: neurobiologic mechanisms.

In apparent contrast to previous results from other labs, we have found that a single exposure to a severe stressor such as immobilization (IMO) caused a long-term desensitization of the hypothalamic-pituitary-adrenal (HPA) response to the homotypic stressor. Because such HPA desensitization was not found in response to heterotypic stressors, it seemed at first that we were describing a habituation process already observed after a single experience with the stressor. However, a more detailed analysis revealed two main properties incompatible with the interpretation of the results in terms of habituation: (1) The intensity of desensitization increases over the course of days to weeks with no additional exposures to the stressor, and (2) the degree of desensitization was greater with more severe stressors. The long-term effects were also observed after a single exposure to a high dose of a systemic stressor such as endotoxin but not after insulin-induced hypoglycemia, suggesting that not all severe systemic stressors can induce such long-term desensitization. Because systemic stressors are known to be processed in specific brain areas and because we have found changes in c-fos mRNA response to the homotypic stressor in some brain areas as a consequence of previous experience with IMO, we hypothesize that some severe stressors do not induce long-term desensitization because they are not processed in brain areas sensitive to previous experience with the stressor. The neurochemical mechanisms involved in the induction of long-term effects on the HPA axis are in process, but our results suggest only a partial role of glucocorticoids and NMDA receptors.

Labelling of poly-L-lysine with 99mTc and evaluation as a possible tracer agent for ventriculography.

A stable 99mTc-labelled compound that is easy to prepare and that is retained for a long period of time in the blood would constitute an ideal replacement for 99mTc-HSA (limited by its rapid diffusion) and 99mTc-erythrocytes (lengthy and risky in vitro labelling) as tracer agent for ventriculography. We investigated whether 99mTc-labelled polymers would be suitable for this purpose. Four types of poly-L-lysine (PL) (Mw 41,000 to 377,000) were used either in underivatized form labelled at pH 12, or derivatized with a varying number of mercaptoacetyl (MA) substituents by reaction with N-hydroxysuccinimidyl-S-acetylmercaptoacetate followed by deprotection with hydroxylamine and labelling at pH 7.5. A high labelling yield was obtained in all cases. HPLC-purified 99mTc-PLs and 99mTc-MA-PLs were evaluated in mice, with 125I-HSA as an internal biological standard. The retention in the blood at 10 minutes and 60 minutes p.i. was not higher than about 30% for any of the tested compounds versus 84% for 125I-HSA, and was only 10% for the smallest 99mTc-labelled PL and MA-PL. Liver uptake was high for the 99mTc-PLs, whereas the 99mTc-MA-PL's were excreted in significant amounts to the urine. It is concluded that 99mTc-labelled poly-L-lysines or polymercaptoacetyl-poly-L-lysines are not suitable as blood pool tracer agents.