Peptides (v.23, #4)

Invertebrate neuropeptides II by R.J. Nachman (603).

The periviscerokinin (PVK) peptide family in insects: evidence for the inclusion of CAP2b as a PVK family member by Christian Wegener; Zsófia Herbert; Manfred Eckert; Reinhard Predel (605-611).
Periviscerokinins (PVKs) are a distinct insect peptide family with unusual distribution in the central nervous system and neurohemal release sites. PVKs were first isolated from the abdominal perisympathetic organs of Periplaneta americana, but can be found in other insect species. Peptides with structural similarity to PVKs have been identified through searches of the Drosophila genome. The cardioacceleratory peptide CAP2b of the hawkmoth Manduca sexta shares close amino acid identity with the PVKs and may thus be included as a structural member of the PVK peptide family. In this review, we provide support for grouping CAP2b as a PVK family member based on published sequences, and new immunocytochemical findings and mass spectrometric data.
Keywords: Neuropeptide; Myotropin; Cardioacceleratory peptide; Drosophila; Cockroach; Perisympathetic organ; Neurohormone;

The association of FMRFamide-related peptides (FaRPs) with the spermatheca of Locusta migratoria was demonstrated using radioimmunoassay and immunohistochemical techniques. The physiological effects of various FaRPs on the neurally evoked contractions of the spermatheca were also examined. FMRFamide-like immunoreactivity (FLI) was demonstrated in processes and cell bodies situated in the VIIIth (terminal) abdominal ganglion. These included an anterior, central and posterior pair of ventral cell bodies positioned near the midline of the ganglion, in addition to two bilaterally paired dorsal cell bodies in the posterior region of the VIIIth abdominal ganglion. Two axons displaying FLI proceed down the ventral ovipositor nerve (VON) and into the receptaculum seminis nerve which innervates the anterior regions of the spermatheca. FLI was also noted in processes on the spermathecal muscle with the highest density occurring on the spermathecal sac and coil duct. FaRPs applied to the spermathecal muscle included GQERNFLRFamide, NFIRFamide, ADDRNFIRFamide, YGGFMRFamide, FMRFamide, ADVGHVFLRFamide and SchistoFLRFamide (PDVDHVFLRFamide). Dose-dependent physiological effects were only noted for FMRFamide, ADVGHVFLRFamide and SchistoFLRFamide. FMRFamide led to a dose-dependent increase in the amplitude of neurally evoked contractions with a threshold of approximately 5 × 10−7 M. SchistoFLRFamide, and ADVGHVFLRFamide, had an inhibitory effect, decreasing the amplitude of neurally evoked spermathecal contractions.
Keywords: FMRFamide-related peptide family; FMRFamide; SchistoFLRFamide; Neurally evoked contractions; Spermatheca; Locusta;

The first member of the periviscerokinin peptide family in Locusta migratoria was identified by post-source decay fragmentation on a MALDI-TOF mass spectrometer using a single neurohemal organ only. The primary sequence of this decapeptide, code-named Lom-PVK, is Ala-Ala-Gly-Leu-Phe-Gln-Phe-Pro-Arg-Val-NH2. Unlike the situation in cockroaches, Lom-PVK is the only abundant periviscerokinin in L. migratoria. It is present in abdominal perisympathetic organs of various species of locusts and grasshoppers. Its myotropic properties, namely to increase the frequency of the contraction of the heart in L. migratoria and stimulate amplitude and tonus of the locust foregut, is reminiscent of the action of Periplaneta-PVKs.
Keywords: Locusta migratoria; Insect; Neuropeptide; Periviscerokinin; Perisympathetic organs; Myotropin;

Coherence between biosynthesis and secretion of insect adipokinetic hormones by Lucien F. Harthoorn; Rob C.H.M. Oudejans; Jacques H.B. Diederen; Dick J. Van der Horst (629-634).
The importance of the process of continuous biosynthesis of locust adipokinetic hormones (AKHs) for the availability of these peptide hormones for release was assessed in vitro by inhibiting this biosynthesis followed by secretory stimulation. Inhibition of the biosynthetic activity for AKHs by brefeldin A caused a considerable inhibition of the AKH release induced by the endogenous crustacean cardioactive peptide (CCAP). After brefeldin A treatment followed by potassium depolarization, CCAP-induced AKH release was completely abolished. In vitro pulse-chase labeling experiments indicated that constitutive secretion from the AKH-producing cells does not occur. It is concluded that AKH secretion involves a regulated release from a relatively small pool of newly formed secretory granules, while older AKH-containing granules appear to be unavailable for release.
Keywords: Adipokinetic hormone (AKH); Peptide hormone; Corpus cardiacum; Neuroendocrine cell; Storage; Release; Biosynthesis; Locusta migratoria (Insecta);

After translation, the AKH I and AKH II precursors form three dimeric constructs prior to further processing into the respective AKHs and three dimeric Adipokinetic Hormone Precursor Related Peptides or APRPs (two homodimers and one heterodimer).By capillary liquid chromatography-tandem mass spectrometry we demonstrate that the APRPs in Locusta migratoria are further processed to form two smaller neuropeptides: DAADFADPYSFL (residue 36 to 47 of the AKH I precursor) and YADPNADPMAFL (residue 34 to 45 of the AKH II precursor). The peptides are designated as Adipokinetic Hormone Joining Peptide 1 (AKH-JP I) and 2 (AKH-JP II) respectively. Within the AKH I and AKH II precursor molecules, the classic KK and RR processing sites separate the AKH-JPs from the AKH I and II respectively. At the carboxyterminus, both AKH-JP I and II are flanked by Tyr-Arg, a cleaving site not described before. Such an unusual cleavage site suggests the presence, in the corpora cardiaca, of specific convertases. The AKH-JP-II does not stimulate lipid release from the fat body nor does it stimulate glycogen phosphorylase activity, both key functions of AKH.
Keywords: Adipokinetic hormone joining peptide; AKH-JP; Peptidomics; Neuropeptide; Nano-LC-MS; Q-TOF; Peptide precusor processing;

Cardioacceleratory effects of Manduca sexta allatotropin in the true armyworm moth, Pseudaletia unipuncta by P.M. Koladich; M. Cusson; W.G. Bendena; S.S. Tobe; J.N. McNeil (645-651).
Manduca sexta allatotropin (Manse-AT), a peptide originally isolated on the basis of its ability to stimulate juvenile hormone (JH) biosynthesis in the tobacco hornworm, is a potent in vitro stimulator of the corpora allata (CA) in Pseudaletia unipuncta (Lepidoptera: Noctuidae). At 10−6M, Manse-AT stimulated in vitro rates of JH biosynthesis by CA of day 0 and 6 adult females 15- and 10-fold respectively. Both Manse-AT and serotonin were also shown to be dose-dependent stimulators of heart rate in day 0, 3 and 6 adult males and females. Furthermore, analysis suggests that there are differences in both resting and Manse-AT-stimulated heart rates depending on age and rearing conditions.
Keywords: Allatostatin; Allatotropin; Cardioacceleratory peptide; Insect heart; Juvenile hormone; Manduca sexta; Pseudaletia unipuncta; Serotonin;

The Manduca allatotropin (Manse-AT) gene is expressed as three mRNAs that differ from each other by alternative splicing. The level of one of these mRNAs (RNA-3) is specifically increased in the nerve cord of last instar larvae that were starved, parasitized, or fed the ecdysteroid agonist RH-5992. Each of these treatments results in reduction of feeding and increased levels of juvenile hormone (JH). The normal decline in JH biosynthesis by the corpora allata does not occur in starved or RH-5992-fed larvae. The increase in RNA-3 levels has the capacity to increase the production of Manse-AT and two related peptides that may be part of the complex response of larvae to nutrient deprivation.
Keywords: Juvenile hormone; Alternative splicing; Neuropeptides; Starvation; Tebufenozide; Parasitism;

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.
Keywords: Insect neuropeptides; Lepidoptera; Terpene biosynthesis; Endocrinology;

The biological activity of diuretic factors in Rhodnius prolixus by V.A Te Brugge; D.A Schooley; I Orchard (671-681).
The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin , however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP2b have been demonstrated to be diuretic factors/hormones.In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards.Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. . As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.
Keywords: Malpighian tubule; Diuresis; cAMP; Hindgut contraction; Neuropeptide;

The role of calcium as a second messenger in the crustacean cardioactive peptide (CCAP)-induced contractions of the locust oviducts was investigated. Incubation of the oviducts in a calcium-free saline containing, a preferential calcium cation chelator, or an extracellular calcium channel blocker, abolished CCAP-induced contractions, indicating that the effects of CCAP on the oviducts are calcium-dependent. In contrast, sodium free saline did not affect CCAP-induced contractions. Co-application of CCAP to the oviducts with preferential L-type voltage-dependent calcium channel blockers reduced CCAP-induced contractions by 32–54%. Two preferential T-type voltage-dependent calcium channel blockers both inhibited CCAP-induced oviduct contractions although affecting different components of the contractions. Amiloride decreased the tonic component of CCAP-induced contractions by 40–55% and flunarizine dihydrochloride decreased the frequency of CCAP-induced phasic contractions by as much as 65%, without affecting tonus. Flunarizine dihydrochloride did not alter the proctolin-induced contractions of the oviducts. Results suggest that the actions of CCAP are partially mediated by voltage-dependent calcium channels similar to vertebrate L-type and T-type channels. High-potassium saline does not abolish CCAP-induced contractions indicating the presence of receptor-operated calcium channels that mediate the actions of CCAP on the oviducts. The involvement of calcium from intracellular stores in CCAP-induced contractions of the oviducts is likely since, an intracellular calcium antagonist decreased CCAP-induced contractions by 30–35%.
Keywords: Calcium; Second messenger; Crustacean cardioactive peptide; Oviducts; Locust;

The salivary glands of the blood-feeding bug, Rhodnius prolixus, are composed of a single epithelial layer of binucleate cells and a double layer of visceral muscle cells surrounding a large secretory cavity. The saliva contains substances which counteract the hemostasis of the host, and injection of saliva into the host is an essential component of successful and efficient gorging.The muscles surrounding the salivary glands of Rhodnius are under polyneuronal control from the salivary nerve projecting out of the hypocerebral ganglion. The amplitude of contractions induced by neural stimulation is dependent upon both intensity and frequency of nerve stimulation.Serotonin and FMRFamide-related peptides (FaRPs) are delivered in the nerve supply to the salivary glands, and both classes of neuroactive chemicals increase frequency and amplitude of phasic contractions in a dose-dependent manner. A member of the FaRP myosuppressin subfamily, however, inhibits contractions. CRF-related and Leucokinin-like peptides are not delivered in the nerve supply but may be present in the hemolymph during feeding. Leucokinin 1 and Zoone DH (a CRF-related peptide) both induce a dose-dependent increase in basal tonus, with phasic contractions superimposed. Zoone DH is more active than Leucokinin 1. Factors are present in the CNS of Rhodnius which mimic the effects of serotonin and the stimulatory peptides.
Keywords: Kinins; CRF-related peptides; FMRFamide-related peptides; Serotonin; Visceral muscle; Insect;

Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide by Geoffrey M. Coast; Janusz Zabrocki; Ronald J. Nachman (701-708).
Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH2) and N-terminal truncated analogs of 4–14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn1, but was reduced > 10-fold after the removal of Thr2. Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys8) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg10, and at 100 μM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg10 was confirmed by comparing dose-response curves for Musdo-K [6–15] and Acheta kinin-V (AFSHWG-NH2) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity.

cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation by Ronald J Nachman; Janusz Zabrocki; Jacek Olczak; Howard J Williams; Guillermo Moyna; A Ian Scott; Geoffrey M Coast (709-716).
The insect kinin neuropeptides have been implicated in the regulation of water balance, digestive organ contraction, and energy mobilization in a number of insect species. A previous solution conformation study of an active, restricted-conformation cyclic analog, identified two possible turn conformations as the likely active conformation adopted by the insect kinins at the receptor site. These were a cisPro type VI β-turn over C-terminal pentapeptide core residues 1–4 and a transPro type I-like β-turn over core residues 2–5, present in a ratio of 60:40. Synthesis and evaluation of the diuretic activity of insect kinin analogs incorporating a tetrazole moiety, which mimics a cis peptide bond, identifies the active conformation as the former. The discovery of a receptor interaction model can lead to the development of potent agonist and antagonist analogs of the insect kinins. Indeed, in this study a tetrazole analog with D stereochemistry has been shown to demonstrate partial antagonism of the diuretic activity of natural insect kinins, providing a lead for more potent and effective antagonists of this critical neuropeptide family. The future development of mimetic agonists and antagonists of insect kinin neuropeptides will provide important tools to neuroendocrinologists studying the mechanisms by which they operate and to researchers developing new, environmentally friendly pest insect control strategies.

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) by hemolymph from larvae of the tomato moth, Lacanobia oleracea was investigated using reversed phase-high performance liquid chromatography (RP-HPLC), and matrix assisted laser desorption ionisation-time of flight mass spectrometry. Metabolism of 1 nmole Manse-AS in diluted hemolymph was rapid, t1/2 = 3.5 min, with a number of products produced. Mass spectrometry of HPLC fractions identified cleavage products, which indicated a sequential degradation of Manse-AS from the N-terminal to Manse-AS (7–15). The most abundant products identified were Manse-AS (5–15), (6–15), and (7–15). These metabolites were synthesized and assayed for biological activity on juvenile hormone (JH) biosynthesis in vitro. All three of the above deletion peptides showed allatostatin activity, but were not as potent as Manse-AS (1–15).
Keywords: Neuropeptide; Allatostatin; Metabolism; Mass spectrometry; Aminopeptidase; Insect; Juvenile hormone synthesis; Manduca sexta; Lacanobia oleracea;

Tachykinin-related peptides (TRP) are widely distributed in the CNS of insects, where they are likely to function as transmitters/modulators. Metabolic inactivation by membrane ecto-peptidases is one mechanism by which peptide signalling is terminated in the CNS. Using locustatachykinin-1 (LomTK-1, GPSGFYGVRamide) as a substrate and several selective peptidase inhibitors, we have compared the types of membrane associated peptidases present in the CNS of four insects, Locusta migratoria, Leucophaea maderae, Drosophila melanogaster and Lacanobia oleracea. A neprilysin (NEP)-like activity cleaving the G-F peptide bond was the major LomTK-1-degrading peptidase detected in locust brain membranes. NEP activity was also found in Leucophaea brain membranes, but the major peptidase was an angiotensin converting enzyme (ACE), cleaving the G-V peptide bond. Drosophila adult head and larval neuronal membranes cleaved the G-F and G-V peptide bonds. Phosphoramidon inhibited both these cleavages, but with markedly different potencies, indicating the presence in the fly brain of two NEP-like enzymes with different substrate and inhibitor specificity. In Drosophila, membrane ACE did not make a significant contribution to the cleavage of the G-V bond. In contrast, ACE was an important membrane peptidase in Lacanobia brain, whereas very little neuronal NEP could be detected. A dipeptidyl peptidase IV (DPP IV) that removed the GP dipeptide from the N-terminus of LomTK-1 was also found in Lacanobia neuronal membranes. This peptidase was a minor contributor to LomTK-1 metabolism by neuronal membranes from all four insect species. In Lacanobia, LomTK-1 was also a substrate for a deamidase that converted LomTK-1 to the free acid form. However, the deamidase was not an integral membrane protein and could be a lysosomal contaminant. It appears that insects from different orders can have different complements of neuropeptide-degrading enzymes. NEP, ACE and the deamidase are likely to be more efficient than the common DPP IV activity at terminating neuropeptide signalling since they cleave close to the C-terminus of the tachykinin, a region essential for maintaining biological activity.
Keywords: Insect tachykinin; Neuronal peptidases; Endopeptidase; Angiotensin converting enzyme; Dipeptidyl peptidase IV; Deamidase;

Enhanced in vivo activity of peptidase-resistant analogs of the insect kinin neuropeptide family by Ronald J. Nachman; Allison Strey; Elwyn Isaac; Nan Pryor; Juan D. Lopez; Jin-Gen Deng; Geoffrey M. Coast (735-745).
The diuretic/myotropic insect kinin neuropeptides, which share the common C-terminal pentapeptide core FX1X2WG-NH2, reveal primary (X2-W) and secondary (N-terminal to F) sites of susceptibility to peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Analogs designed to enhance resistance to tissue-bound peptidases, and pure insect neprilysin and ACE, demonstrate markedly enhanced in vivo activity in a weight gain inhibition assay in H. zea, and strong in vivo diuretic activity in the housefly (M. domestica). The peptidase-resistant insect kinin analog pQK(pQ)FF[Aib]WG-NH2 demonstrates a longer internal residence time in the housefly than the native muscakinin (MK), and despite a difference of over 4 orders of magnitude in an in vitro Malpighian tubule fluid secretion assay, is equipotent with MK in an in vivo housefly diuretic assay. Aminohexanoic acid (Ahx) is shown to function as a surrogate for N-terminal Lys, while at the same time providing enhanced resistance to aminopeptidase attack. Peptidaese-resistant insect kinin analogs demonstrate enhanced inhibition of weight gain in larvae of the agriculturally destructive corn earworm moth. Potent peptidase resistant analogs of the insect kinins, coupled with an increased understanding of related regulatory factors, offer promise in the development of new, environmentally friendly pest insect control measures.

Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta by E. Lee; A. Lange; I. Orchard; M. Fusé; S.S. Tobe; W.G. Bendena; B.C. Donly (747-756).
Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.
Keywords: FMRFamide-related peptide; Baculovirus; Neuropeptide; Insect;

Drosophila melanogaster TDVDHVFLRFamide (DMS), SDNFMRFamide, and pEVRFRQCYFNPISCF (FLT) represent three structurally distinct peptide families. Each peptide decreases heart rate albeit with different magnitudes and time-dependent responses. DMS and FLT are expressed in the crop and decrease crop motility; however, SDNFMRFamide expression and effect on the crop has not been reported. These data suggest the peptides have different physiological roles. The peptides have non-overlapping expression patterns in neural tissue, which suggests different mechanisms regulate their synthesis and release. The structures, expression patterns, and activities of the myotropins suggest they have important but different roles in biology and different signaling pathways.
Keywords: Allatostatin; Dromyosuppressin; Flatline; FMRFamide; Heart rate; Mas-AS; Myosuppressin; SDNFMRFamide;

A putative SchistoFLRFamide receptor in CNS membrane preparations of Locusta migratoria was characterized by cold competition binding and kinetic binding assays using [125I][Y1]SchistoFLRFamide ([125I]YDVDHVFLRFamide) as a radioligand. Binding to this site was saturable, specific, reversible, and of high-affinity. Data fit to a single-site binding model by non-linear regression (r2 = 0.99) estimated Kd = 1.73 ± 0.45 × 10−9 M and Bmax = 49.0 ± 12.2 fmol · mg−1 tissue. Total binding of [125I][Y1]SchistoFLRFamide to membrane preparations was reduced in the presence of GTPγS, an indication that the putative receptor is G protein-coupled. Structure-activity studies determined that the minimum sequence required for binding was HVFLRFamide. Other aspects of the ligand receptor interaction were also examined.
Keywords: Insect; Neuropeptides; FMRFamide-related peptides; Competition binding assay; GTPγS;

Characterization of a functional neuropeptide F receptor from Drosophila melanogaster by Stephen F. Garczynski; Mark R. Brown; Ping Shen; Thomas F. Murray; Joe W. Crim (773-780).
Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner (IC50 = 65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC50 = 51 nM). Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF.
Keywords: Insect; Diptera; Drosophila; G protein-coupled receptor; Neuropeptide F; Neuropeptide Y;

The genomic organization of the open reading frame of the red pigment concentrating hormone gene in the blue crab Callinectes sapidus by Francisco Martı́nez-Pérez; Jesús Valdés; Samuel Zinker; Hugo Aréchiga (781-786).
The open reading frame (ORF) of the gene for the precursor of the octapeptide Red Pigment Concentrating Hormone (RPCH) from the blue crab Callinectes sapidus was cloned by PCR with oligonucleotides targeted to the initiation and the end of the translation coding sequences. A 272 bp intron was characterized between nucleotides 343 and 344 of the reported cDNA, present in the region coding for the last amino acids of the precursor related peptide of RPCH. The intron genomic structure here described is similar to that reported for the gene coding for the Adipokinetic Hormone (AKH) of the grasshopper Schistocerca nitans.
Keywords: Crab; Crustacean; Gene; Intron; Invertebrate; Neurohormone; Neuropeptide; Peptide; Red Pigment Concentrating Hormone;

We identified a Drosophila melanogaster gene encoding a peptide that dramatically decreases spontaneous muscle contractions and, correspondingly, named the peptide flatline (FLT). This gene consisted of 4 exons and was cytologically localized to 32D2–3. Processing of a predicted 122 amino acid precursor would release pEVRYRQCYFNPISCF that differs from Manduca sexta allatostatin (Mas-AST) by one amino acid, Y4→F4. FLT does not act as an allatostatin. In situ tissue hybridization further suggests FLT is a novel brain-gut peptide and specifically, the measured activity indicates that it is a potent myotropin. Despite its profound myotropic effect, pupae injected with FLT eclosed.
Keywords: Brain-gut peptide; Drosophila; Juvenile hormone regulation; Myotropin;

A new, p -carborane containing analog of tyrosine, 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran (12)-12-yl]alanine, was prepared from protected 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran (12)-12-yl]propionic acid in five steps using Oppolzer’s sultam methodology for asymmetric hydroxyamination as the key step. The tyrosine mimetic can function as a hydrophobic surrogate for tyrosine residues in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs. As an amino acid, insertion of the mimic is not limited to the N-terminus but can replace a tyrosine residue at any position within a peptide sequence.
Keywords: p-carboranes; Insect peptides; Synthesis; Tyrosine analog;

An analog of the insect pyrokinin/PBAN class of neuropeptides, which features a 2-amino7-bromofluorene attached to the carboxy-terminal bioactive core of the insect pyrokinin/PBAN class of neuropeptides (Phe-Thr-Pro-Arg-Leu-NH2), via a succinnic acid linker, was tested in adult H. virescens moths. This analog was found to induce pheromone production when injected into or applied topically to moths. Topical application of as much as 1 nmol of the analog to moths induced production of significant amounts of pheromone for only 1–2 h, whereas injection of 500 pmol induced pheromone production for up to 20 h. All insects died within 24 h after injection of 500 pmol of the analog. Mortality studies indicated that the LD50 for the analog was 0.7 pmol when injected. A non-pyrokinin/PBAN peptide analog formed by attachment of 2-amino-7-bromofluorene to Ala-Ala-Arg-Ala-Ala-NH2 (via the succinnic acid linker) did not induce mortality when injected at 1 nmol. Similarly no mortality was found when up to 2 nmol of an analog containing a non-brominated fluorene ring, formed by attachment of 9-fluoreneacetic acid to Phe-Thr-Pro-Arg-Leu-NH2, was injected into moths. The data indicated that both the bromine and active core of the pyrokinin neuropeptides (Phe-Thr-Pro-Arg-Leu-NH2) were critical for a specific toxic action and suggested that the brominated analog poisoned the moths by interacting with pyrokinin receptors.
Keywords: Insect neuropeptides; Pyrokinin; PBAN; Pheromone production; Toxicity;

Insulin-related peptides and their conserved signal transduction pathway by Ilse Claeys; Gert Simonet; Jeroen Poels; Tom Van Loy; Linda Vercammen; Arnold De Loof; Jozef Vanden Broeck (807-816).
The ‘insulin superfamily’ is an ancient category of small, structurally related proteins, such as insulin, insulin-like growth factors (IGF) and relaxin. Insulin-like signaling molecules have also been described in different invertebrates, including nematodes, mollusks, and insects. They initiate an evolutionary conserved signal transduction mechanism by binding to a heterotetrameric, membrane-spanning receptor tyrosine kinase. Recent physiological and genetic studies have revealed that, in different metazoans, the insulin signaling pathway plays a pivotal role in the regulation of a variety of interrelated, fundamental processes, such as metabolism, growth, reproduction and aging.
Keywords: Aging; Caenorhabditis elegans; Cell size; Drosophila melanogaster; Growth; Hormone; Invertebrate; Metabolism; Obesity; Peptide; Receptor; Reproduction;