Peptides (v.27, #3)

Contents List (v-vi).

Novel excitatory neuropeptides isolated from a prosobranch gastropod, Thais clavigera: The molluscan counterpart of the annelidan GGNG peptides by F. Morishita; H. Minakata; K. Takeshige; Y. Furukawa; T. Takata; O. Matsushima; S.T. Mukai; A.S.M. Saleuddin; T. Horiguchi (483-492).
The GGNG peptides are excitatory neuropeptides identified from earthworms, leeches and polychaeta. Two structurally related peptides were purified and characterized from a mollusk, Thais clavigera (prosobranch gastropod). The peptides designated as Thais excitatory peptide-1 (TEP-1) (KCSGKWAIHACWGGN-NH2) and TEP-2 (KCYGKWAMHACWGGN-NH2) are pentadecapeptides having one disulfide bond and C-terminal GGN-NH2 structures, which are shared by most GGNG peptides. TEP augmented the motilities of Thais esophagus and penial complex. TEP-like immunoreactivity is distributed in both the neurons of the central nervous system and nerve endings in the penial complex. Thus, the involvement of TEP in the contraction of the digestive and reproductive systems is suggested. Substitution of amino acids in TEP revealed that two tryptophan residues in TEP are important for maintaining bioactivity.
Keywords: HPLC; Purification; Reproduction; Imposex; Organotin;

Cloning and characterization of a third isoform of corazonin in the honey bee Apis mellifera by Peter Verleyen; Geert Baggerman; Inge Mertens; Tim Vandersmissen; Jurgen Huybrechts; Alfons Van Lommel; Arnold De Loof; Liliane Schoofs (493-499).
The precursor of the insect hormone corazonin has been cloned from the honey bee Apis mellifera. The precursor predicts a novel isoform of corazonin, pQTFTYSHGWTNamide, which was confirmed by tandem mass spectrometry. Although Apis corazonin differs only by a glutamine/threonine substitution from [His7]-corazonin, it is considerably less active in the dark color inducing assay on albino locusts. Whole mount fluorescence immunohistochemistry of the central nervous system of the honey bee showed a pattern similar to the ones described for other insects. Four neurons of the lateral protocerebrum project axons towards the retrocerebral complex. It is unlikely that Apis corazonin is present in all hymenopteran species since the presence of this peptide could not be demonstrated by means of mass spectrometry in the retrocerebral complex of the red wood ant Formica rufa and the wasp Vespula saxonica. Instead, we found masses corresponding with [Arg7]- and [His7]-corazonin respectively, suggesting that some of the corazonin isoforms originated late during evolution in different insect orders.
Keywords: Apis mellifera; Drosophila melanogaster; Hymenoptera; Mass spectrometry; Corazonin; Neuropeptide precursor;

A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius by Manuel B. Aguilar; Leticia Lezama-Monfil; María Maillo; Heriberto Pedraza-Lara; Estuardo López-Vera; Edgar P. Heimer de la Cotera (500-505).
A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68 Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42 Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I–III, II–IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0 nmol) into mice, peptide sr5a caused depressed behavioral activity.
Keywords: Conidae; Cone snail; Conus spurius; Conotoxin; Hydrophobic; T-1-conotoxin;

Amino acid sequence and biological activity of a γ-conotoxin-like peptide from the worm-hunting snail Conus austini by Alejandro Zugasti-Cruz; María Maillo; Estuardo López-Vera; Andrés Falcón; Edgar P. Heimer de la Cotera; Baldomero M. Olivera; Manuel B. Aguilar (506-511).
A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVγCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif –γCCS–, which has only been found in the γ-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other γ-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.
Keywords: Conoidea; Conidae; Cone snail; Conus austini; Conotoxins; Vermivorous; Worm-hunting; γ-Conotoxin;

The neuropeptide profiles and diversity of the brain and retrocerebral organs (corpora cardiaca–corpora allata; CC–CA) of adult workers of the honey bee Apis mellifera carnica (dark European strain) were investigated using a combination of HPLC and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with post-source decay (PSD) and collision-induced dissociation (CID) fragmentation. Using evidence from genomic sources, including BLAST searches of the honey bee genome, comparisons with other species and de novo sequencing by PSD and CID fragmentation, a total of 13 mass ions could be assigned to peptides predicted from the A. mellifera genomic database. Peptides positively identified were A. mellifera tachykinin-related peptides 3 and 4 (APMGFQGMRa; APMGFYGTRa) and leucomyosuppressin (pEDVDHVFLRFa). Peptides tentatively identified were A. mellifera tachykinin-related peptides 2 and 5 (ALMGFQGVRa; ARMGFHGMRa), A. mellifera allatostatins 2, 3 and 4 (GRDYSFGLa; RQYSFGLa; GRQPYSFGLa), A1-SIFamide (AYRKPPFNGSIFa), Q1-leucomyosuppressin (QDVDHVFLRFa) and A. mellifera pyrokinins PK 1, PK 2 and Q1-PK 2 (TSQDITSGMWFGPRLa; pEITQFTPRLa; QITQFTPRLa). Allatostatins, tachykinin-related peptides and A1-SIFamide were not detected in CC–CA extract, which appears to contain predominantly leucomyosuppressin, Q1-leucomyosuppressin, PK 1, PK 2, Q1-PK 2 and some unidentified masses. No ion signal was detected that would correspond to the hypertrehalosaemic peptide (=Manse-AKH), which has been isolated from the Italian race of the honey bee (A. mellifera ligustica), but not from A. mellifera carnica.
Keywords: Peptidomics; Insect; Hymenoptera; Allatostatin; Leucomyosuppressin; Pyrokinin; Tachykinin-related peptide; SIFamide;

Identification of PVK/CAP2b neuropeptides from single neurohemal organs of the stable fly and horn fly via MALDI-TOF/TOF tandem mass spectrometry by Ronald J. Nachman; William K. Russell; Geoffrey M. Coast; David H. Russell; J. Allen Miller; Reinhard Predel (521-526).
MALDI-TOF/TOF tandem mass spectrometry has been applied to determine the complete sequences of the PVK/CAP2b neuropeptides in the stable fly Stomoxys calcitrans and horn fly Haematobia irritans, insect pests of livestock. This peptidomic analysis of single neurohemal organ preparations allows the unambiguous assignment of internal Leu/Ile positions not distinguishable by previous mass spectrometric techniques. The sequences are as follows: Stoca-PVK/CAP2b-1, AGGASG L YAFPRVa; Stoca-PVK/CAP2b-2, NAK L YPVPRVa; and Haeir-PVK/CAP2b-1, AGGASG L YAFPRVa; Haeir-PVK/CAP2b-1, NAK L YPMPRVa. Both Stoca-PVK/CAP2b-1 and -2 stimulate Malpighian tubule fluid secretion in the stable fly, with EC50 values between 3 and 11 nM. The identification of these novel neuropeptides adds to our knowledge of the peptidomes of flies, and can aid in the development of neuropeptide-based control strategies of these insect pests.
Keywords: MALDI-TOF/TOF mass spectrometry; Insect; Neuropeptide; Periviscerokinin; CAP2b; Stomoxys calcitrans; Haematobia irritans; Peptidomics;

Five native pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L9]Neb-PVK-2, [I9]Neb-PVK-2) identified in the neuropeptidome of the flesh fly Neobellieria bullata were compared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I9]Neb-PVK-2 proved inactive, whereas [L9]Neb-PVK-2 demonstrated only weak activity. In contrast, both Neb-PK-1 and Neb-PK-2 demonstrated potent threshold doses, with Neb-PK-2 about 10-fold more active than Neb-PK-1. Analysis of neuromuscular activity during pupariation using a tensiometric technique demonstrates that the two native Neb-PKs accelerate the onset of immobilization and cuticular shrinkage more than motor programs associated with retraction of the anterior segments and longitudinal body contraction. It was further determined that the sensitivity of various components of the pupariation process to these peptides decreases in the following order: immobilization > cuticular shrinkage > motor program for anterior retraction > motor program for longitudinal contraction ≅ tanning of cuticle of the newly formed puparium. A paradoxical situation was observed whereby the motor programs of pupariation are temporally dissociated from actual morphogenesis of the puparium. The tensiometric data suggest that the most likely candidate for a primary pupariation factor is Neb-PK-2, rather than Neb-PK-1.
Keywords: Flesh fly; Neobellieria bullata; Diptera; Pupariation; Pyrokinins; Control; Behavioral patterns; Cuticle tanning;

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L. by Akira Yamanaka; Miwa Adachi; Hiroshi Imai; Terumasa Uchiyama; Moeko Inoue; A.T.M. Fayezul Islam; Chisato Kitazawa; Katsuhiko Endo (534-538).
Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head–thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG2,3–AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG2,3–AG1–7 complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG2,3–AG1–7 complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.
Keywords: Butterfly; Papilio xuthus; Orange-Pupa-Inducing Factor (OPIF); Pupal coloration; Polymorphism;

Degradation profile of [His7]-corazonin in the hemolymph of the desert locust Schistocerca gregaria by Tim Vandersmissen; Bruno Hoste; Geert Baggerman; Jurgen Huybrechts; Arnold De Loof; Patrick Chaltin; Paul Proost; Michael Breuer (539-548).
Degradation of the neuropeptide [His7]-corazonin, a key hormone in phase transition in locusts was studied using [3H][His7]-corazonin, RP-HPLC and mass spectrometry. After 4 h incubation, 50 and 75% of [His7]-corazonin could still be found in hemolymph of gregarious and solitarious Schistocerca gregaria, respectively. Under in vivo conditions the half-life was 30 min. These results are in contrast to many other neuropeptides that usually have half lives of a few minutes. The peptide is cleaved first by an endopeptidase, either just before or after the Tyr residue at position 5. Next, the C-terminal degradation fragments are further degraded by a dipeptidyl-peptidase, whereas the N-terminal fragments are further broken down one amino acid at a time. In addition, [Dopa5][His7]-corazonin was detected. Upon synthesis, this unexpected molecular modification turned out to be biologically active in bringing about cuticular melanization.
Keywords: Corazonin; Locust; Phase transition; Endopeptidase; Peptidyl-dopa;

Proctolin-like immunoreactivity (PLI) was widely distributed in the locust, Locusta migratoria, within the central, peripheral and stomatogastric nervous systems, as well as the digestive system and retrocerebral complex. Proctolin-like immunoreactivity was observed in cells and processes of the brain and all ganglia of the ventral nerve cord. Of interest, PLI was found in the lateral neurosecretory cells, which send axons within the paired nervi corporis cardiaci II (NCC II) to the corpus cardiacum (CC). The CC contained extensive processes displaying PLI, which continued on within the paired nervi corporis allata (NCA) to the paired corpora allata (CA) where the axons entered and branched therein. The frontal and hypocerebral ganglia of the stomatogastric nervous system contained PLI within processes, resulting in a brightly staining neuropile. Each region of the gut contained PLI in axons and processes of varying patterns and densities. The paired ingluvial ganglia contained PLI, including an extensively stained neuropile and immunoreactive axons projecting through the nerves to the foregut. The hindgut contained PLI within longitudinal tracts, with lateral projections originating from the 8th abdominal ganglion via the proctodeal nerve. The midgut contained PLI in a regular latticework pattern with many varicosities and blebs. No difference in PLI in cells and processes of the central nervous system (CNS) was found between males and females.
Keywords: Immunohistochemistry; Stomatogastric nervous system; Neuropeptide; Nervous system; Gut; Corpus cardiacum; Corpus allatum;

Proctolin: A possible releasing factor in the corpus cardiacum/corpus allatum of the locust by Lisa Clark; Jin Rui Zhang; Stephen Tobe; Angela B. Lange (559-566).
The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10−8  M and maximal release at about 10−7  M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (<35 pmol h−1 per CA). In contrast, proctolin did not alter the synthesis and release of JH from the cockroach CA. These results suggest that proctolin may act as a releasing factor for AKHs and JH in the locust but does not act as a releasing factor for JH in the cockroach.
Keywords: Adipokinetic hormone; Juvenile hormone; Cockroach; Diploptera punctata; Locusta migratoria; Immunohistochemistry; Bioassay; Lipid mobilization;

The levels of three alternatively spliced mRNAs from the Manduca sexta allatotropin (Manse-AT) gene were determined following physiological manipulations during the larval, pupal and adult stages; starvation of larvae, induction of pupal diapause and adult mating experience. The juvenile hormone biosynthetic activity of the corpora allata (CA) was also determined in starved larvae and in mated and unmated females. Starvation of early fifth instar larvae specifically increased the amount of one Manse-AT mRNA that is predicted to encode Manse-AT and two related peptides, Manse-ATL-I and -II. The normal rapid decrease in the activity of the CA in last instar larvae was not observed in starved insects which maintained a relatively high rate of JH biosynthesis for at least 3 days. Diapause induction resulted in a small increase in one Manse-AT mRNA, but levels were much lower compared to those observed in larvae or adults. During the first 4 days of adult life, Manse-AT mRNA levels were not changed as a result of mating. However, in mated females, the rate of JH biosynthesis gradually increased, in sharp contrast to the relatively low level of CA activity seen in virgin females. These observations suggest the elevated activity of the CA in mated females is not simply due to the increased level of Manse-AT mRNA.
Keywords: Neuropeptides; Allatotropin; Juvenile hormone; Starvation; Mating; Diapause; Manduca sexta;

Structural studies of Drosophila short neuropeptide F: Occurrence and receptor binding activity by Stephen F. Garczynski; Mark R. Brown; Joe W. Crim (575-582).
Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids were clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs.
Keywords: Insect; Drosophila; G protein-coupled receptor; Neuropeptide F; Neuropeptide Y;

Neuropeptides are important messenger molecules in invertebrates, serving as neuromodulators in the nervous system and as regulatory hormones released into the circulation. Understanding the function of neuropeptides will require the integration of genetic, biochemical, physiological and behavioral information. The advent of DNA microarrays and bioinformatic databases provides a wealth of data describing the expression profiles of thousands of genes during biological processes. One such array catalogs the developmental patterns of gene expression during the metamorphic transformation of the Drosophila midgut. We have mined the data from this experiment to explore changes of expression in genes coding for known neuropeptides, peptide hormones, and their receptors during the metamorphosis of the midgut. We found small but significant changes in the expression of the peptides diuretic hormone, FGLa-type allatostatins, myoinhibiting peptide, ecdysis-triggering hormone, drosokinin and the burs subunit of bursicon, as well as the receptors DAR-2, NPFR1, ALCR-2, Lkr and DH-R. Just as advances have been made in understanding the molecular basis of invertebrate neuropeptide action by analysis of genome projects, data mining of gene expression databases can help to integrate molecular, biochemical and physiological knowledge of biological processes.
Keywords: Insect peptides; Microarrays; Differential expression; Midgut; Metamorphosis;

The hemolymph of invertebrates contains factors that facilitate clotting as a defense mechanism for injury. However, the clotting process may impair the measurement of hormone titers by interfering with the extraction of peptides. Using hemolymph from freshwater crayfish, our results demonstrate that hemolymph clotting appears to reduce both the amount of an endogenous peptide(s) (Dippu-AST 11-like) extracted from hemolymph, as well as the amount of spiked peptide tracer ([125I]-Dippu-AST 11) recovered from hemolymph. The efficacy of peptide extraction from hemolymph was improved by collecting the hemolymph into a variety of different media prior to hemolymph coagulation. Hemolymph samples collected into media containing the anticoagulant, citrate, had the highest amount of endogenous Dippu-AST 11-like peptide extracted as well as the highest percent recovery of spiked tracer.
Keywords: Anticoagulant; Citrate; Insect; Crustacean; Hemolymph; Peptide; Allatostatin;

Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia by Scott F. Cummins; Amy E. Nichols; Catherine H. Schein; Gregg T. Nagle (597-606).
The water-borne protein attractin is a potent sex pheromone involved in forming and maintaining mating and egg-laying aggregations in the marine mollusk Aplysia. Binary blends of attractin and either enticin, temptin, or seductin, three other Aplysia protein pheromones, stimulate mate attraction. The four pheromones are thought to act in concert during egg-laying. The new data presented here show that: (1) the water-borne odor of non-laying Aplysia brasiliana further increases the attractiveness of attractin and of eggs in T-maze bioassays. This suggests that individual Aplysia release additional factors that enhance the effects of attractin, enticin, temptin, and seductin during egg-laying; (2) the N-terminal region of enticin aligns well with the conserved epidermal growth factor (EGF)-like domain of mammalian reproductive proteins known as fertilins, which may mediate intercellular adhesion interactions between eggs and sperm; (3) temptin, according to fold recognition servers, may also have an EGF-like fold. Enticin and temptin also have conserved metal binding sequences that may play a role in their signaling behavior. These results suggest that aspects of mammalian egg–sperm interactions (fertilins) may have evolved from pheromonal signaling mechanisms. We also review the structure, expression, localization, release, and behavioral actions of attractin, enticin, temptin, and seductin.
Keywords: Aplysia; Attractin; Enticin; Temptin; Seductin; Fertilin; Fibrillin; Pheromone;