A major development has been the development of 8 antisera raised against the gastrin/cholecystokinin (related neuropeptide drosulphankinin (DSK)II). These antisera have been used to map the distribution of immunoreactive cells in Calliphora vomitoria and compare them with neurones that are immunoreactive to mammalian CCK. The amino acid sequences of the 2 gastrin/CCK and DSK peptides are very similar at the C-terminus. Despite this sequence similarity, the cytological evidence is that there may be both a DSK homologue and a more mammalian certain cells share immunoreactivities to both antisera, other antisera. This is an intriguing story and will require further study before it is resolved. Further collaborative studies on this topic have been planned.
Work has been carried out on the isolation and amino acid sequencing of 14 neuropeptides designated the calli phenylalanine methionine arginine phenylalanine amides (calliFMRFamides) that are related to gastrin/cholecystokinin (CCK) peptides at the C-terminus (-plenylalanine-methionine-arginine-phenlalanine-amide compared with tryptophan-methionine-aspartic acid-phenylalanine-amide). They were isolated from extracts of the nervous system of Calliphora vomitoria using a radioimmunoassay (RIA) against tyrosine-glycine-glycine-phenylalanine-methionine-arginine- phenylalanine-oxide and a more nonspecific arginine-phenylalanine-amide RIA. It was hoped that the CCK-peptides or drosulphakinin (DSK)- peptides would have been isolated using these procedures but so far this has not happened. The newly development antisera against DSK II will, however, now be used on the fractions that remain from these various extractions and it is hoped that by this means the peptides can be isolated. Arising from the gastrin/CCK/DSK/FMRFamide peptide isolation studies has come an unexpected finding that may prove of considerable importance in the future development of innovative insect control methods. Thus, by sequencing 1 particular ultraviolet (UV) peak that was weakly immunoreactive in the RIAs, the first dipteran neuropeptide with allatostatic properties has been identified (ie the ability to inhibit the release of Juvenile Hormone (JH) from the corpus allatum). The peptide is homologous with the previously identified allatostations of the cockroach Diploptere punctata. Subsequently, the RIA has been developed against the C vomitoria callatostatin a further 4 members of this neutopeptide family have been isolated, 1 of which is a more potent inhibitor of JH production int he cockroach that any peptide possessed by the cockroach itself. The real significance and potential importance of these JH inhibitory peptides lies in the fact that JH is vital to the process of oocyte formation and, without it, insects are unable to produce eggs. Clearly, if it is possible to inhibit the production and release of JH, then the potential for breaking the reproductive cycle exists.
A major success has recently been achieved with the cloning and sequencing of the drosulphakinin (DSK) related gene in Calliphora vomitoria. The genomic deoxyribonucleic acid (DNA) library of C vomitoria, produced from ripe eggs proved to be prefect for screening purposed and, using standard techniques, the Calliphora gene of the prohormone was isolated and the putative peptide sequences compared with Drosophila metanogaster and Lucilia cuprina. The conservation of amino acid sequences in 3 dipteran species in this way suggests the possibility of an important functional significance and future studies will be directed towards elucidating this.
Another major success has been the cloning and sequencing of the calli phenylalanine methionine arginine phenylalanine amide (calliFMRFamide) gene. This work represents one of the first instances in an insect species, or indeed in any species, where the same research group has isolated a particular group of peptides (the 14 calliFMRFamides) and also has achieved the cloning and sequencing of the gene responsible for the peptides. Analysis of the prohormone shows that there are a total of 18 putative calliFMRFamides in tandem, with 15 different molecular species. One of the peptides identified was not present on the gene suggesting, perhaps, that another FMRFamide gene may exist.
Conformation activity relationships of the calli phenylalanine methionine arginine phenylalanine amides (calliFMRFamides) has been vigorously pursued. The specific objectives of the work have been to establish why it is that only 3 out of a group of 5 very similar nonapeptides (out of the calliFMRFamides) are able to induce fluid secretion from the isolated salivary gland of the blowfly Calliphora vomitoria. Computer modelling techniques have been used to make a theoretical evaluation of the relationships between the primary structures of these particular 5 calliFMRFamides, their conformation in aqueous solution and their sialagogic activities. These studies, which are continuing, combine molecular modelling with molecular mechanics/simulations using AMBER on the CONVEX C3840 super computer at the University of London Computing Centre. So far the active serine-proline-serine-glutamine-aspartic acid-phenylalanine-methionine-arginine-phenylalanine-amide was compared with the nonactive arginine-proline-glycine-glutamine-aspartic acid-phenylalanine-methionine-arginine-phenylalanine-amide. The results have demonstrated very satisfactorily that this group of nonapeptides, which vary only at positions 1 and 3, are active if they possess type I beta turns between residues 1 and 4 and between 3 and 7, but are inactive if they have other types of turn such as beta II. Other physiological parameters that have been investigated include actions of the CCK/DSK-related and calliFMRFamide peptides on the gut and heart.
Neuropeptides belonging to the gastrin/cholecystokinin family will be studied in the blowfly, Calliphora vomitoria. Several experimental approaches will be used to give an integrated view. Morphological studies will identify the cellular localisation of the peptides, biochemical studies will provide information on their chemical identity; molecular biological studies (the recombinant DNA approach) will be used to identify gene and peptide sequences; and physiological studies will assess the role of the peptides in aspects of reproduction and feeding.
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Funding SchemeCSC - Cost-sharing contracts
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