Browsing by Author "Gäde, Gerd"
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- ItemOpen AccessFunctional characterisation of the hypertrehalosaemic hormone from the Indian stick insect Carausius morosus: metabolic and myotropic studies(2017) Katali, Ottilie Kandiwapa Hasiike; Marco, Heather G; Gäde, GerdNeuropeptides of the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family are well known as regulators for many physiological processes in insects, notably energy metabolism, and a possible role in myostimulation. The Indian stick insect Carausius morosus contains two members of this family, hypertrehalosaemic hormone I and II (Carmo-HrTH-I and -II). Both these are decapeptides and they differ only at position 8, where the tryptophan of Carmo-HrTH-I is C‐mannosylated. It is known that Carmo-HrTHs increase the carbohydrate (trehalose) concentration in the haemolymph via a G protein-coupled receptor. The current study seeks to identify which part of the HrTH amino acid sequence is necessary to interact with the receptor on the fat body of C. morosus to trigger a response (hypertrehalosaemia) eventually leading to the release of carbohydrates into the haemolymph. In addition, the role of Carmo-HrTHs in stimulating the heart rate through myostimulation was also investigated. Two biological assays were used to assess the potencies of various analogues, in comparison to that of the native peptides: (1) the carbohydrate-mobilizing assay assessed the increase in levels of carbohydrates; and (2) the semi-exposed heart assay assessed the increase in heart rates. The current study confirmed that both Carmo-HrTH-I and -II are capable of increasing the haemolymph carbohydrates in ligated stick insects. The results of the current study revealed, for the first time, that these peptides also similarly stimulate the heart rate of the stick insect. Thus, Carmo-HrTH-II was used as a lead peptide in the current study on which various naturally-occurring AKH peptides and systematically altered analogues were based. The selected naturally-occurring AKH peptides had a single or double amino acids replacement in comparison to Carmo-HrTH-II and some were octapeptides. Each systematically altered analogue of the native Carausius HrTH II had a single amino acid replaced with alanine. Additionally, two analogues that lacked the N-terminal pyroglutamate residue or had a free threonine acid at the C-terminus instead of an amide were also tested. The results showed that the N- or C- terminal modified analogues have no hypertrehalosaemic activity in C. morosus and are also incapable of increasing the heart rate of this insect as high as Carmo-HrTH-II. This suggests that the blocked termini are important features, for both peptide protection and receptor binding. The structural requirements of C. morosus receptor(s) for Carmo-HrTHs appear to be very specific. The receptor(s) do not accept octapeptides and only four out of the fourteen decapeptides elicited at least 46% of the biological activity as compared to the native peptide and the rest (ten) were not active. This implies that the HrTHs receptor (s) for C. morosus do not tolerate the replacement of most single amino acids. In the heart assay, known cardio-stimulatory peptides were applied to the semi-exposed heart of C. morosus to establish the potential extent of cardioexcitation. The results of this study revealed that crustacean cardioactive peptide and proctolin are capable of increasing the heart beat rate of the stick insect more than the AKH/RPCH peptides. It was also shown that the stick insect heart beat rate can be inhibited by octopamine. This is the first study to investigate, in detail, the importance of structural features of the hypertrehalosaemic hormones of the Phasmatodea insects, and is therefore an important contribution to designing environmentally friendly insect-specific pesticides.
- ItemOpen AccessThe gonadotropin-releasing hormone (GnRH) system: a comparison between breeding and non-breeding naked mole rats (Heterocephalus glaber)(2010) Smith, Caitlin; Marco, Heather G; Gäde, GerdNeuropeptides are well known to govern numerous biological functions and are found in all phyla studied to date. Probably the best known neuroendocrine system is the hypophyseal-portal system found in vertebrates, and one of the functions of this system is to mediate reproduction. Mammalian reproduction is controlled by a hormonal cascade which begins in distinct brain regions, namely the hypothalamus and the pituitary gland. Gonadotropin-releasing hormone (GnRH) is a neuropeptide typically produced in the hypothalamus. It is the key neuropeptide for initiating this cascade, and without it, reproduction cannot occur. Naked mole rats (Heterocephalus glaber) have a rigid social hierarchy. The "queen" is the most dominant female and is the only female who breeds. All aspects of reproduction are suppressed in other females in the colony: these "subordinates" are in a prepuberty-like state as they do not ovulate or display breeding behaviours. They are, however, not infertile, and are capable of rising to the breeding position. Since GnRH is the "master hormone" of reproduction, this study investigates its role in the socially-induced suppression of reproduction in female H. glaber. Brains of breeding (n = 7) and non-breeding (n = 5) female naked mole rats were compared to determine any differences in brain size, particularly in regions related to GnRH production. Noteworthy morphological and physiological transformations accompany the change from subordinate to dominant social status, including a significant increase in body length (Mann Whitney U test; p = 0.005, U = 0.000), body mass (Mann Whitney U test; p = 0.009, 1.000) and pituitary width and length (Mann Whitney U test; p = 0.028, U = 0.500 and p = 0.018, U = 0.000, respectively). Since little is known about the GnRH system in H. glaber, this study used immunocytochemistry to identify the distribution and abundance of GnRH neurons in the brains of both breeding and non-breeding females. GnRH neurons were found in the median eminence of the hypothalamus and in the anterior pituitary of both queens and subordinates, however in the brain of queen (n = 7) naked mole rats, there is a significantly larger area of immunoreactivity in comparison to the subordinate (n = 5) brain tissue (Mann Whitney U = 4.000, p = 0.030). This suggests that, in , subordinates, GnRH is inhibited at the level of production. The amino acid structure of the form of GnRH found in the brain of the naked mole rat is currently unknown, therefore a pilot study was carried out, using synthetic mammalian GnRH (mGnRH), mouse brain tissue and naked mole rat pituitaries and hypothalami, to examine and modify (where necessary) the methodologies used for neuropeptide extraction, purification and identification. A limited number of naked mole rats were available as source tissue (n = 4), therefore this study also tested whether it is possible to extract and purify an unknown neuropeptide from only a few mammalian samples. Training for reverse-phase liquid chromatography (RP-HPLC) was achieved by practicing the necessary methods with crude extracts prepared from stick insect (Carausius morosus) corpora cardiaca, which also served to compare vertebrate and invertebrate neuroendocrine systems. Synthetic mGnRH was used to demonstrate repeatability of the protocol and to set up suitable conditions for elution of mGnRH: mGnRH elutes at â 12 min when a solvent gradient of 32 % - 47 % B is applied. Synthetic mGnRH was also used to establish the amount of peptide required for accurate identification of GnRH by antigenicity tests (ELISA) and mass spectrometry. However, when extracts of mouse or naked mole rat brain matter were applied to this system, purification of GnRH was unconvincing as either there was insufficient material, or, some endogenous factor was masking the GnRH. Future studies would benefit from using molecular techniques as they require smaller amounts of source tissue. Alternatively, a larger amount of source tissue would be necessary in order to proceed with biochemical studies. While the impaired production of GnRH in subordinate naked mole rats seems to be linked to their prepuberty like state, it is unlikely that GnRH is the sole factor involved. Many other hormones (such as kisspeptin, gonadotropin-inhibiting hormone and neuropeptide Y) may influence GnRH and sexual maturity, and future studies would benefit from a multi-layered approach to investigate suppression of reproduction in naked mole rats.
- ItemOpen AccessHormonal control of flight metabolism in Odonata?(1995) Janssens, Markus Peter-Erik; Gäde, GerdMembers of the AKHIRPCH family of peptides were identified in corpora cardiaca of the dragonfly Anax imperator (Anisoptera: Aeshnidae), Orthetrumjuliajalsum (Anisoptera: Libellulidae) and the damselflies Pseudagrion inconspicuum and Ischnura senegalensis (Zygoptera: Coenagrionidae). After isolation ofthe peptides by reversed phase high performance liquid chromatography, the primary structures were established by Edman sequencing and mass spectrometry (Ani-AKH: pGlu-Val-Asn-Phe-Ser-Pro-Ser-TrpNH 2 ), (Lia-AKH: pGlu-Val-Asn-Phe¬ Thr-Pro-Ser- TrpNH 2 ) and (Psi-AKH: pGlu- Val-Asn-Phe- Thr-Pro-Gly- TrpNH 2 ). One corpus cardiac of A. imperator contains about 40 pmol Ani-AKH, O. julia 19-24 pmol Lia¬ AKH and P. inconspicuum about 2.4 pmol Psi-AKH. Injection of Ani-AKH (3.4 pmol) increased the concentration of haemolymph lipids in A. imperator. Lia-AKH (l pmol) similarly had an adipokinetic effect in 0. julia. Psi-AKH (I pmol) had an adipokinetic effect, as well as a small hyperglycaemic effect in P. inconspicuum. The AKH peptides of other Odonata were investigated. In the suborder Anisoptera, Ani-AKH was identified in representatives of the Aeshnidae, Cordulegasteridae, and possibly the Corduliidae. Lia¬ AKH was identified in representatives of the Libellulidae and Gomphidae. In the suborder Zygoptera, Psi-AKH was identified in representatives of the families Chlorolestidae, Lestidae and Chlorocyphidae, and possibly the Calopterygidae and Protoneuridae. Classification of Odonata according to their flight behaviour as "perchers" or "fliers" is supported by parameters of energy metabolism. Lipid metabolism seems to have a greater importance in fliers than perchers. The lipid concentration in the haemolymph is highest in the flier A. imperator, intermediate in the percher 0. julia and lowest in the percher P. inconspicuum. There are indications that mitochondria isolated from flight muscles of A. imperator may have a higher capacity for lipid oxidation than 0. julia. The contribution of carbohydrates to flight metabolism seems to be more important in perchers than in fliers. The concentration of carbohydrates in the haemolymph is highest in P. inconspicuum, intermediate in O. julia and lowest inA. imperator. The maximal activity of phosphofructokinase (a rate-limiting enzyme of glycolysis) is higher in the percher, O. julia, than in the flier, A. imperator. The lipid concentration in the haemolymph is higher than that of the carbohydrates in O. julia, A. imperator and P. inconspicuum. Palmitoyl-carnitine is oxidised at high rates by isolated mitochondria from flight muscles of O. julia andA. imperator, similar to Locusta migratoria. Lipid is the major fuel utilised during flight in O. julia. Carbohydrates (in the haemolymph) and proline (in the haemolymph and flight muscles) are utilised as minor fuels. It is concluded that the processes of lipid metabolism provide the major source of energy during flight in Odonata. The AKH peptides seem to play a role in regulating lipid mobilisation during flight in Odonata.
- ItemOpen AccessIn Silico Screening for Pesticide Candidates against the Desert Locust Schistocerca gregaria(Multidisciplinary Digital Publishing Institute, 2022-03-07) Jackson, Graham E; Gäde, Gerd; Marco, Heather GAdipokinetic hormone (AKH) is one of the most important metabolic neuropeptides in insects, with actions similar to glucagon in vertebrates. AKH regulates carbohydrate and fat metabolism by mobilizing trehalose and diacylglycerol into circulation from glycogen and triacylglycerol stores, respectively, in the fat body. The short peptide (8 to 10 amino acids long) exerts its function by binding to a rhodopsin-like G protein-coupled receptor located in the cell membrane of the fat body. The AKH receptor (AKHR) is, thus, a potential target for the development of novel specific (peptide) mimetics to control pest insects, such as locusts, which are feared for their prolific breeding, swarm-forming behavior and voracious appetite. Previously, we proposed a model of the interaction between the three endogenous AKHs of the desert locust, Schistocerca gregaria, and the cognate AKHR (Jackson et al., Peer J. 7, e7514, 2019). In the current study we have performed in silico screening of two databases (NCI Open 2012 library and Zinc20) to identify compounds which may fit the endogenous Schgr-AKH-II binding site on the AKHR of S. gregaria. In all, 354 compounds were found to fit the binding site with glide scores < −8. Using the glide scores and binding energies, 7 docked compounds were selected for molecular dynamic simulation in a phosphatidylcholine membrane. Of these 7 compounds, 4 had binding energies which would allow them to compete with Schgr-AKH-II for the receptor binding site and so are proposed as agonistic ligand candidates. One of the ligands, ZINC000257251537, was tested in a homospecific in vivo biological assay and found to have significant antagonistic activity.
- ItemOpen AccessInsights into the Activation of a Crustacean G Protein-Coupled Receptor: Evaluation of the Red Pigment-Concentrating Hormone Receptor of the Water Flea Daphnia pulex (Dappu-RPCH R)(2021-05-10) Jackson, Graham E.; Gäde, GerdThe validation of a previously developed model of the interaction between the red pigment-concentrating hormone of Daphnia pulex and its cognate receptor (Jackson et al., IJBM 106, 969–978, 2018) was undertaken. Single amino acid replacements, noticeably an Ala scan, of the ligand, Dappu-RPCH, were docked to the receptor, and the binding energies calculated and compared to the one with Dappu-RPCH. As a second step, the same molecules were docked using molecular dynamics (MD) in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane. Changes in binding energy were compared to previous results on in vitro receptor activation (Marco et al., Sci. Rep. 7, 6851, 2017). Residue scanning and MD simulations both gave comparable results for binding energy. For most mutants, there was a good inverse correlation between in vitro activity and binding. There were, however, exceptions; for example: [Ala4]Dappu-RPCH bound as tightly as the cognate ligand but had little activity. This seeming discrepancy was explained when the MD data were analyzed in detail, showing that, although [Ala4]Dappu-RPCH had multiple interactions with the receptor accounting for the high binding energy, the interacting residues of the receptor were quite different to those of Dappu-RPCH. The MD calculations show clearly that the strong binding affinity of the ligand to the receptor is not sufficient for activation. Interaction of the binding of the ligand to two residues of the receptor, Ser 155 and Gln 237, is also essential. A comparison of our computational results with the experimental results of Marco et al. and comparison with the extensive data on GnRH supports the validity of our Dappu-RPCH R model.
- ItemOpen AccessNeuropeptide hormones from the eyestalks of Jasus Lalandii(2000) Marco, Heather G; Gäde, Gerd; Cook, Peter; Brandt, Wolf FThe X-organ sinus gland complex, situated in the eyestalks of decapod crustaceans, are known to be a source of a variety of neuropeptide hormones that regulate a number of diverse physiological processes. This neuroendocrine complex was investigated in 3 crustacean species, viz. the European shore crab Carcinus maellas, and 2 spiny lobster species Jasus lalandii and Panulirus homarus by means of tissue immunocytochemistry and an enzyme-linked immunosorbent assay (ELISA). Positive immunoreactions, associated with the X-organ - sinus gland system only, were obtained with antisera raised against crustacean hyperglycaemic hormone (cHH) of the American lobster (Homarus americanus), the Mexican crayfish (Procambarus bouvieri) and the edible crab (Cancer pagurus), as well as with antisera raised against vitellogenesis-inhibiting hormone (VIH) of the H. americanus and moult-inhibiting hormone (MIH) of C. pagurus. This is the first time that the immunolocalisation of these 3 hormones have been studied in a single crustacean species. The chief results of this comparative immunocytochemical study showed that (1) neuropeptide hormones of the shore crab and the 2 spiny lobster species were sufficiently homologous in primary structure to be recognised by the heterologous antisera, thus, an indication of conserved peptide structures across the species and infraorder boundaries; (2) preabsorbed complexes of purified peptides and antisera from the edible crab did not produce any immunoreactions in tissue immunocytochemistry, nor in ELISA, thus, indicating the specificity of the anti-cHH and anti-MIH sera; (3) the anti-VIH serum demonstrated the ability to bind epitopes on cHH and MIH peptides and is, thus, not a specific antiserum in this study; (4) there is co-localisation of cHH, MIH, VIH immunoreactivity in the eyestalk neuroendocrine complexes of all 3 species studied which suggests that the different peptide hormones can be synthesized in the same neuronal cell bodies. This co-localisation of neuropeptides in the eyestalk of J lalandii was confirmed by a double-staining immunoflourescence experiment, and finally (5) immunoreactivity of antisera raised against cHH of H. americanus and MIH of C. pagurus was associated with distinct and unique peak fractions, following reverse-phase high pressure liquid chromatographic (RP-HPLC) separation of sinus gland extracts from J. lalandii. A total of 6 neuropeptide hormones belonging to the cHH/MIH/VIH peptide family were isolated, functionally characterised and sequenced from extracts of sinus glands from the South African west coast rock lobster, Jasus lalandii. This is the first complete report on these peptides from any species belonging to the Palinuridae infraorder.
- ItemOpen AccessRole and distribution of astaxanthin in spiny lobster, Jasus lalandii(2006) Matumba, Tshifhiwa Given; Marco, Heather G; Gäde, Gerd; Auerswald, LThe occurance and distribution of astaxanthin in tissues of the spiny lobster, Jasus lalandii was investigated. The concentration of astaxanthin was quantified in the exoskeleton, haemolymph, muscles, gonads and hepatopancreas as well as in egg parcels from berried females during the moult as well as the reproductive cycles. Astaxanthin was the dominant carotenoid in all tissues, but small amounts of other carotenoids were detected. Exoskeleton, ovaries and extrude egg parcels had significantly higher astaxanthin concentration than haemolymp, muscles and hepatopancreas. This distribution of astaxanthin was found in both captive and free-living spiny lobsters. Free-living spiny lobsters generally had higher astaxanthin concentration than the captive spiny lobsters although the data is not statistically significant in all tissues investigated.