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Calliphora vomitoria

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Alan Thorpe – 1st expert on this subject based on the ideXlab platform

  • identification of the dipteran leu callatostatin peptide family characterisation of the prohormone gene from Calliphora vomitoria and lucilia cuprina
    Regulatory Peptides, 1996
    Co-Authors: Peter D East, Hanne Duve, Karen Tregenza, Alan Thorpe

    Abstract:

    Abstract The prohormone gene encoding the Leu-callatostatin peptides has been isolated from a Calliphora vomitoria genomic DNA library and its homologue was cloned from genomic and cDNA libraries of another blowfly species, Lucilia cuprina. Gene and prohormone structure and organisation are essentially identical in the two species. The Leu-callatostatin gene consists of at least 3 exons. The prohormone is encoded on exons two and three and the two blocks of putative Leu-callatostatin peptides are carried on separate exons. It is 180 amino-acids long, begins with a short signal peptide and contains two blocks of tandemly arranged Leu-callatostatin peptides separated by an acidic spacer region. The prohormone contains 5 copies of the C-terminal sequence -YXFGL characteristic of the Leu-callatostatin family. Complete endoproteolytic processing at all possible pairs of basic amino acids would generate 5 different Leu-callatostatin octapeptides. Two larger Leu-callatostatins could be released if processing was not complete at two of the sites. None of the 3 peptides encoded in the first block was identified in previous purification studies of the callatostatin peptides. The second block, located at the carboxyl end of the prohormone, contains two peptide sequences identical to the previously isolated Leu-callatostatins 1 and 4. The absence of independent copies of Leu-callatostatins 2 and 3 on the prohormone establishes that endoproteolytic cleavage of the precursor does not invariably proceed to completion and that Leu-callatostatin 2 must be derived by N-terminal processing of the parent peptide Leu-callatostatin 1. Reverse transcriptase PCR analysis of mRNA from brain and midgut, the two major sites of Leu-callatostatin expression, shows that the prohormone sequence at these two sites is identical, ruling out the possibility that different populations of peptides are expressed in these two tissues as a result of alternative RNA splicing.

  • identification of the dipteran leu callatostatin peptide family the pattern of precursor processing revealed by isolation studies in Calliphora vomitoria
    Regulatory Peptides, 1996
    Co-Authors: Hanne Duve, Peter D East, Alan G Scott, Anders H Johnsen, Joseluis Maestro, Alan Thorpe

    Abstract:

    Abstract Information from the Leu-callatostatin gene sequences of the blowflies Calliphora vomitoria and Lucilia cuprina was used to develop antisera specific for the variable post-tyrosyl amino-acid residues Ser, Ala and Asn of the common Leu-callatostatin C-terminal pentapeptide sequence −YXFGL-NH 2 . Radioimmunoassays based on these antisera were used to purify peptides from an extract of 40 000 blowfly heads. Five neuropeptides of the Leu-callatostatin family were identified. Three have a seryl residue in the post-tyrosyl position. Two of these are octapeptides that differ only at the N-terminal residue; NRPYSFGL-NH 2 and ARPYSFGL-NH 2 , whilst the third is the heptapeptide derived by N-terminal trimming; RPYSFGL-NH 2 . Two octapeptides in which X is Ala and Asn were also identified; VERYAFGL-NH 2 and LPVYNFGL-NH 2 . The latter peptide is derived by processing at the internal dibasic site of a putative heneicosapeptide encoded by the DNA. These findings stress the necessity to have putative structures verified at the peptide level. Potent, reversible inhibitory effects on the spontaneous contractile activity of the blowfly rectum were recorded for ARPYSFGL-NH 2 (monophasic dose-response curve with an IC 50 = 10 fM) and for LPVYNFGL-NH 2 (biphasic dose-response curve with IC 50 values of approximately 1 fM and 1 nM). It is suggested that regulation of gut motility in insects, rather than an allatostatic function, may represent an ancestral and universal function of the allatostatins. One of the reasons for the large number of members of the Leu-callatostatin family appears to be in the provision of an integrated form of gut motility control, with different peptides controlling specific regions of the gut.

  • the sulfakinins of the blowfly Calliphora vomitoria
    FEBS Journal, 1995
    Co-Authors: Hanne Duve, Alan Thorpe, Alan G Scott, Anders H Johnsen, Jens F Rehfeld, Eric R Hines, Peter D East

    Abstract:

    The nonapeptide, Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2 was isolated from heads of the blowfly Calliphora vomitoria. Designated callisulfakinin I, the peptide is identical to the earlier known drosulfakinin I of Drosophila melanogaster and to neosulfakinin I of Neobellieria bullata. It belongs to the sulfakinin family, all known members of which (from flies, cockroaches and locusts) have the C-terminal heptapeptide sequence Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2. The callisulfakinin gene of C. vomitoria was cloned and sequenced. In addition to callisulfakinin I, the DNA revealed a coding sequence for the putative tetradecapeptide, Gly-Gly-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-NH2, callisulfakinin II. However, this peptide was not identified in the fly head extracts. Confocal laser scanning immunocytochemical studies with antisera raised against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster, Asp-Gln-Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2, revealed only four pairs of sulfakinin neurones in the brain of C. vomitoria and no others anywhere else in the neural, endocrine or gut tissues. In situ hybridisation studies with a digoxigenin-labelled sulfakinin gene probe (from the blowfly Lucilia cuprina) also revealed only four pairs of neurones in the brain. The perikarya of two pairs of cells are situated medially in the caudo-dorsal region, close to the roots of the ocellar nerve. The other perikarya are slightly more posterior and lateral. Although it has been suggested by several authors that the insect sulfakinins are homologous to the vertebrate peptides gastrin and cholecystokinin, such arguments (based essentially on C-terminal structural similarities) do not take account of important differences in the C-terminal tetrapeptide, His-Met-Arg-Phe-NH2 in the sulfakinins, compared with Trp-Met-Asp-Phe-NH2 in gastrin and cholecystokinin. Furthermore, whereas the sulfakinin neurones of C. vomitoria are small in number and have a very specialised location, a greater number of cells throughout the nervous system react positively to gastrin/cholecystokinin antisera. Chromatographic profiles of the present study also revealed peaks of gastrin/cholecystokinin-immunoreactive material separate from the sulfakinin peptides. This evidence suggests that the insect and vertebrate peptides may not necessarily be homologous.

Hanne Duve – 2nd expert on this subject based on the ideXlab platform

  • identification of the dipteran leu callatostatin peptide family characterisation of the prohormone gene from Calliphora vomitoria and lucilia cuprina
    Regulatory Peptides, 1996
    Co-Authors: Peter D East, Hanne Duve, Karen Tregenza, Alan Thorpe

    Abstract:

    Abstract The prohormone gene encoding the Leu-callatostatin peptides has been isolated from a Calliphora vomitoria genomic DNA library and its homologue was cloned from genomic and cDNA libraries of another blowfly species, Lucilia cuprina. Gene and prohormone structure and organisation are essentially identical in the two species. The Leu-callatostatin gene consists of at least 3 exons. The prohormone is encoded on exons two and three and the two blocks of putative Leu-callatostatin peptides are carried on separate exons. It is 180 amino-acids long, begins with a short signal peptide and contains two blocks of tandemly arranged Leu-callatostatin peptides separated by an acidic spacer region. The prohormone contains 5 copies of the C-terminal sequence -YXFGL characteristic of the Leu-callatostatin family. Complete endoproteolytic processing at all possible pairs of basic amino acids would generate 5 different Leu-callatostatin octapeptides. Two larger Leu-callatostatins could be released if processing was not complete at two of the sites. None of the 3 peptides encoded in the first block was identified in previous purification studies of the callatostatin peptides. The second block, located at the carboxyl end of the prohormone, contains two peptide sequences identical to the previously isolated Leu-callatostatins 1 and 4. The absence of independent copies of Leu-callatostatins 2 and 3 on the prohormone establishes that endoproteolytic cleavage of the precursor does not invariably proceed to completion and that Leu-callatostatin 2 must be derived by N-terminal processing of the parent peptide Leu-callatostatin 1. Reverse transcriptase PCR analysis of mRNA from brain and midgut, the two major sites of Leu-callatostatin expression, shows that the prohormone sequence at these two sites is identical, ruling out the possibility that different populations of peptides are expressed in these two tissues as a result of alternative RNA splicing.

  • identification of the dipteran leu callatostatin peptide family the pattern of precursor processing revealed by isolation studies in Calliphora vomitoria
    Regulatory Peptides, 1996
    Co-Authors: Hanne Duve, Peter D East, Alan G Scott, Anders H Johnsen, Joseluis Maestro, Alan Thorpe

    Abstract:

    Abstract Information from the Leu-callatostatin gene sequences of the blowflies Calliphora vomitoria and Lucilia cuprina was used to develop antisera specific for the variable post-tyrosyl amino-acid residues Ser, Ala and Asn of the common Leu-callatostatin C-terminal pentapeptide sequence −YXFGL-NH 2 . Radioimmunoassays based on these antisera were used to purify peptides from an extract of 40 000 blowfly heads. Five neuropeptides of the Leu-callatostatin family were identified. Three have a seryl residue in the post-tyrosyl position. Two of these are octapeptides that differ only at the N-terminal residue; NRPYSFGL-NH 2 and ARPYSFGL-NH 2 , whilst the third is the heptapeptide derived by N-terminal trimming; RPYSFGL-NH 2 . Two octapeptides in which X is Ala and Asn were also identified; VERYAFGL-NH 2 and LPVYNFGL-NH 2 . The latter peptide is derived by processing at the internal dibasic site of a putative heneicosapeptide encoded by the DNA. These findings stress the necessity to have putative structures verified at the peptide level. Potent, reversible inhibitory effects on the spontaneous contractile activity of the blowfly rectum were recorded for ARPYSFGL-NH 2 (monophasic dose-response curve with an IC 50 = 10 fM) and for LPVYNFGL-NH 2 (biphasic dose-response curve with IC 50 values of approximately 1 fM and 1 nM). It is suggested that regulation of gut motility in insects, rather than an allatostatic function, may represent an ancestral and universal function of the allatostatins. One of the reasons for the large number of members of the Leu-callatostatin family appears to be in the provision of an integrated form of gut motility control, with different peptides controlling specific regions of the gut.

  • the sulfakinins of the blowfly Calliphora vomitoria
    FEBS Journal, 1995
    Co-Authors: Hanne Duve, Alan Thorpe, Alan G Scott, Anders H Johnsen, Jens F Rehfeld, Eric R Hines, Peter D East

    Abstract:

    The nonapeptide, Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2 was isolated from heads of the blowfly Calliphora vomitoria. Designated callisulfakinin I, the peptide is identical to the earlier known drosulfakinin I of Drosophila melanogaster and to neosulfakinin I of Neobellieria bullata. It belongs to the sulfakinin family, all known members of which (from flies, cockroaches and locusts) have the C-terminal heptapeptide sequence Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2. The callisulfakinin gene of C. vomitoria was cloned and sequenced. In addition to callisulfakinin I, the DNA revealed a coding sequence for the putative tetradecapeptide, Gly-Gly-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-NH2, callisulfakinin II. However, this peptide was not identified in the fly head extracts. Confocal laser scanning immunocytochemical studies with antisera raised against the synthetic undecapeptide C-terminal fragment of drosulfakinin II from D. melanogaster, Asp-Gln-Phe-Asp-Asp-Tyr(SO3)-Gly-His-Met-Arg-Phe-NH2, revealed only four pairs of sulfakinin neurones in the brain of C. vomitoria and no others anywhere else in the neural, endocrine or gut tissues. In situ hybridisation studies with a digoxigenin-labelled sulfakinin gene probe (from the blowfly Lucilia cuprina) also revealed only four pairs of neurones in the brain. The perikarya of two pairs of cells are situated medially in the caudo-dorsal region, close to the roots of the ocellar nerve. The other perikarya are slightly more posterior and lateral. Although it has been suggested by several authors that the insect sulfakinins are homologous to the vertebrate peptides gastrin and cholecystokinin, such arguments (based essentially on C-terminal structural similarities) do not take account of important differences in the C-terminal tetrapeptide, His-Met-Arg-Phe-NH2 in the sulfakinins, compared with Trp-Met-Asp-Phe-NH2 in gastrin and cholecystokinin. Furthermore, whereas the sulfakinin neurones of C. vomitoria are small in number and have a very specialised location, a greater number of cells throughout the nervous system react positively to gastrin/cholecystokinin antisera. Chromatographic profiles of the present study also revealed peaks of gastrin/cholecystokinin-immunoreactive material separate from the sulfakinin peptides. This evidence suggests that the insect and vertebrate peptides may not necessarily be homologous.

Ian R Dadour – 3rd expert on this subject based on the ideXlab platform

  • Blowflies & nicotine: an entomotoxicology study
    , 2020
    Co-Authors: Paola A Magni, Marco Pazzi, Marco Vincenti, Eugenio Alladio, M. Brandimarte, Ian R Dadour

    Abstract:

    This research describes the development and validation of a suitable analytical method, based on GC-MS, to detect nicotine in larvae, pupae, empty puparia and adults of blowfly Calliphora vomitoria L. (Diptera: Calliphoridae). Furthermore, the effects on the blowfly survival and growth rate were examined when reared on substrates spiked with three concentrations of nicotine, sufficient to cause death in humans.

  • development and validation of an hplc ms ms method for the detection of ketamine in Calliphora vomitoria l diptera calliphoridae
    Journal of Forensic and Legal Medicine, 2018
    Co-Authors: Paola A Magni, Marco Pazzi, Marco Vincenti, Jessica Droghi, Ian R Dadour

    Abstract:

    Abstract Entomotoxicology is a branch of forensic entomology that studies the detection of drugs or other toxic substances from insects developing on the decomposing tissues of a human corpse or animal carcass. Entomotoxicology also investigates the effects of these substances on insect development, survival and morphology to provide an estimation of the minimum time since death. Ketamine is a medication mainly used for starting and maintaining anesthesia. In recent years ketamine has also been used as a recreational drug, and occasionally as a sedating drug to facilitate sexual assault. In both activities, it has resulted in several deaths. Furthermore, ketamine has been also implicated in suspicious deaths of animals. The present research describes for the first time the development and validation of an analytical method suited to detect ketamine in larvae, pupae, empty puparia, and adults of Calliphora vomitoria L. (Diptera: Calliphoridae), using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This research also considers the effects of ketamine on the survival, developmental rate and morphology (length and width of larvae and pupae) of C. vomitoria. The larvae were reared on liver substrates homogeneously spiked with ketamine concentrations consistent with those found in humans after recreational use (300 ng/mg) or allegedly indicated as capable of causing death in either humans or animals (600 ng/mg). The results demonstrated that (a) HPLC-MS/MS method is applicable to ketamine detection in C. vomitoria immatures, not adults; (b) the presence of ketamine at either concentration in the food substrate significantly delays the developmental time to pupal and adult instar; (d) the survival of C. vomitoria is negatively affected by the presence of ketamine in the substrate; (e) the length and width of larvae and pupae exposed to either ketamine concentration were significantly larger than the control samples.

  • development and validation of a method for the detection of α and β endosulfan organochlorine insecticide in Calliphora vomitoria diptera calliphoridae
    Journal of Medical Entomology, 2018
    Co-Authors: Paola A Magni, Marco Pazzi, Marco Vincenti, Valerio Converso, Ian R Dadour

    Abstract:

    Entomotoxicology studies employ analytical methods and instrumentation to detect chemical substances in carrion insects feeding from the decomposing tissues. The identification of such chemicals may determine the cause of death and may be used for the estimation of the minimum time since death. To date, the main focus of entomotoxicological studies has been the detection of drugs, whereas little information concerns the effects of pesticides on blowflies. Pesticides are generally freely available and more affordable than drugs but they can also be a home hazard and an accessible candidate poison at a crime scene. A QuEChERS extraction method followed by Gas chromatography–mass spectrometry (GC-MS) analysis was developed for the detection of α- and β-endosulfan (organochlorine insecticide and acaricide) in Calliphora vomitoria L. (Diptera: Calliphoridae) and validated. Furthermore, the effects of endosulfan on the morphology, development time and survival of the immature blowflies were investigated. Larvae were reared on liver substrates homogeneously spiked with aliquots of endosulfan corresponding to the concentrations found in body tissues of humans and animals involved in endosulfan poisoning. Results demonstrated that the combination of QuEChERS extraction and GC-MS provide an adequate methods to detect both α- and β-endosulfan in blowfly immatures. Furthermore, the presence of α- and β-endosulfan in the food source 1) prevented C. vomitoria immatures reaching the pupal instar and, therefore, the adult instar at high concentrations, 2) did not affect the developmental time of blowflies at low concentrations 3) affected the size of immatures only at high concentrations, resulting in significantly smaller larvae.