Graham J. Goldsworthy

Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria

In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust, Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.

A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera).

A new member of the AKH/RPCH family was isolated and identified from the corpora cardiaca of the firebug Pyrrhocoris apterus. The peptide was isolated in a single step by reversed phase HPLC and the structure deduced from the multiple MS (MS(N)) electrospray mass spectra and amino acid analysis as that of an octapeptide with the sequence pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH(2): this sequence was confirmed by synthesis. The synthetic peptide induced lipid mobilisation and stimulated locomotory activity in macropterous females. This peptide, designated as Pyrrhocoris apterus adipokinetic hormone (Pya-AKH), is the first identified adipokinetic hormone described in a representative species of the suborder Heteroptera.

Adipokinetic hormone enhances laminarin and bacterial lipopolysaccharide-induced activation of the prophenoloxidase cascade in the African migratory locust, Locusta migratoria

Lom-AKH-I enhances the activation in vivo of prophenoloxidase in the haemolymph of the African migratory locust, Locusta migratoria, in response to challenge with laminarin. AKH does not influence the speed or initial magnitude of the phenoloxidase response to laminarin, but prolongs the period of activation of the enzyme in a dose-dependent manner. Injections of preparations of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase in vivo, but co-injection of Lom-AKH-I with commercial preparations of LPS from Klebsiella pneumoniae, Escherichia coli, or Shigella flexneri (but not one from Pseudomonas aeroginosa) results in dose-dependent increases in the levels of phenoloxidase that persist in the haemolymph for several hours. It is argued that the effects of AKH on phenoloxidase activation in locusts described here are, at least in part, related directly to changes in lipid metabolism brought about by the hormone.

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.

Interactions between the endocrine and immune systems in locusts

Abstract. The prophenoloxidase cascade in the haemolymph of mature adult Locusta migratoria migratorioides (R & F) is activated in response to injection of laminarin, a β‐1,3 glucan. Co‐injection of adipokinetic hormone‐I (Lom‐AKH‐I) and laminarin prolongs the activation of the enzyme in a dose‐dependent manner. However, injections of bacterial lipopolysaccharide (LPS) do not activate prophenoloxidase unless AKH is co‐injected, when there is a dose‐dependent increase in the level of phenoloxidase that persists in the haemolymph for several hours. Even when AKH is co‐injected, the highest levels of phenoloxidase activity are always greater after injection of laminarin than after LPS, and these two immunogens must activate the prophenoloxidase cascade by quite distinct pathways. In the present study, interactions between the endocrine and immune systems were examined with respect to activation of prophenoloxidase and the formation of nodules: injection of LPS induces nodule formation in adult locusts. With LPS from Pseudomonas aeruginosa, nodules form exclusively in dense accumulations in the anterior portion of the abdomen on either side of the dorsal blood vessel associated with the dorsal diaphragm. However, with LPS from Escherichia coli, fewer nodules are formed but with a similar distribution, except that occasionally some nodules are aligned additionally on either side of the ventral nerve cord. Co‐injection of Lom‐AKH‐I with LPS from either bacteria stimulates greater numbers of nodules to be formed. This effect of coinjection of AKH on nodule formation is seen at low doses of hormone with only 0.3 or 0.4 pmol of Lom‐AKH‐1, respectively, increasing the number of nodules by 50%. Injections of octopamine or 5‐hydroxytryptamine do not mimic either of the actions of Lom‐AKH‐I described here. Co‐injection of an angiotensin‐converting enzyme inhibitor, captopril, reduces nodule formation in response to injections of LPS but has no effect on the activation of phenoloxidase. Co‐injection of an inhibitor of eicosanoid synthesis, dexamethasone, with LPS influences nodule formation (with or without AKH) in different ways according to the dose of dexamethasone used, but does not affect activation of prophenoloxidase. Eicosanoid synthesis is important for nodule formation, but not for the activation of the prophenoloxidase cascade in locust haemolymph.

Circulating Levels of Locusta Diuretic Hormone: The Effect of Feeding

A radioimmunoassay was developed using 125I-labeled-[TyrO]Locusta-DH and polyclonal antibodies raised against Locusta-DH (29-46). The assay had a detection limit of 50 pM, and displayed limited cross-reactivity for other CRF-related peptides, but not for unrelated peptides. About 60% of the total immunoreactive material in locust hemolymph was attributable to Locusta-DH. The circulating level of diuretic hormone increases fivefold in fed insects, sufficient to stimulate primary urine production, and is correlated with the duration of the meal. This is consistent with the role of Locusta-DH in the control of postfeeding diuresis.

STRUCTURES, ASSAYS AND RECEPTORS FOR LOCUST ADIPOKINETIC HORMONES

This review is concerned mainly with the adipokinetic hormones (AKHs) of locusts: their molecular conformations, actions and functions and the development of microfiltration assays in vitro. The physiological significance of having multiple hormones with overlapping actions whose efficacy changes during development is discussed in relation to the possibility that these reflect variations in populations of receptors and/or the pharmacokinetics of the peptides. The involvement of second messengers in the transduction mechanism of AKHs is reviewed, and we describe hormone-induced changes of intracellular calcium in single dispersed fat body cells. The structure activity relationships of the three locust AKHs and a number of analogues with variations at the N- and C-termini are discussed. A number of areas are identified where there are gaps in our understanding of these hormones, and some of these will be the focus of our future research.

Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed.

Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

BackgroundGranulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined.ResultsUsing an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations.ConclusionOverall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae.