Peptides (v.30, #3)

Contents List (v-vi).

Invertebrate neuropeptides IX by Ronald J. Nachman (445-448).

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC–MALDI-TOF MS by Steven J. Husson; Bart Landuyt; Thomas Nys; Geert Baggerman; Kurt Boonen; Elke Clynen; Marleen Lindemans; Tom Janssen; Liliane Schoofs (449-457).
Neuropeptides are important signaling molecules that function in cell–cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.
Keywords: Neuropeptide; Peptidomics; Mass spectrometry; MALDI-TOF MS; flp; nlp;

Structural characterization of five post-translationally modified isomorphs of a novel putative δ-conotoxin from the vermivorous snail Conus delessertii from the Mexican Caribbean Sea by Manuel B. Aguilar; Artemisa Flores-Torres; César V.F. Batista; Andrés Falcón; Estuardo López-Vera; Edgar P. Heimer de la Cotera (458-466).
A novel peptide, de7b, was isolated from the venom of Conus delessertii, a worm-hunting species collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry: it contains 28 amino acids, including six Cys residues. Peptide de7b is the second, O-conotoxin-like peptide isolated from the venom of this species, and it exists in different post-translationally modified isomorphs, some of which contain gamma-carboxy-glutamate (γ) and/or 4-hydroxy-proline (O) at positions 4, 7, and/or 14. Its primary structure is DCI(P/O)GG(E/γ)NCDVFR(O/P)YRCCSGYCILLLCA, with molecular masses varying from 3078.6 to 3154.6 Da, depending on the number and kind of modified amino acid residues. Peptide de7b shows significant sequence identity with several O-conotoxins purified and biologically characterized from molluscivorous and piscivorous cone snails of the Indo-Pacific region, the tropical Atlantic and Eastern Pacific Oceans, especially with the δ-conotoxins but also with the ω-conotoxins from molluscivorous species, which suggests that it might affect voltage-gated Na+ or Ca2+channels. Peptide de7b has 32% sequence identity with putative γ-conotoxin de7a, previously characterized from the same species; both peptides contain the same number of amino acid residues and of non-Cys residues between the pairs of consecutive Cys residues. However, these peptides have charge differences at seven positions within the N-terminal half indicating that they might have distinct molecular targets that remain to be identified.
Keywords: Conoidea; Conidae; Conus delessertii; O-conotoxin; γ-Carboxy-glutamate; 4-Hydroxy-proline;

A novel peptide, pal9a, was purified from the venom duct extract of the turrid snail, Polystira albida (superfamily Conoidea, family Turridae), collected in the Gulf of Mexico. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry. Turritoxin pal9a contains 34 amino acid residues, including 6 Cys residues arranged in the pattern C-C-C-C-C-C (framework IX, where “-” represents one or more non-Cys amino acids), which characterizes the P-conotoxins. Peptide pal9a is the first P-conotoxin-like turritoxin characterized from a member of family Turridae of the Western Atlantic. The primary structure of turritoxin pal9a, NVCDGDACPDGVCRSGCTCDFNVAQRKDTCFYPQ-nh2 (-nh2, amidated C-terminus; calculated monoisotopic mass, 3679.48 Da; experimental monoisotopic mass, 3678.84 Da), shows variable degrees of low sequence similarity with framework IX-toxins from turrid (three species of Lophiotoma, and four species of Gemmula), terebrid (Hastula hectica), and Conus species of the Indo-Pacific (C. textile, C. gloriamaris, C. amadis, and C. litteratus) and of the Western Atlantic (C. regius). During the comparison of peptide pal9a with the other framework IX-toxins known to date, we realized that, in general, these peptides are hydrophilic, acidic compounds that have not been found in the fish-hunting Conus species studied thus far; we also found support for the notion that they may belong to several distinct gene superfamilies, even those from the same species. Given the broad distribution of framework IX-toxins within superfamily Conoidea, it will be interesting to identify the still-unknown molecular targets of P-conotoxins, P-conotoxin-like turritoxins, and P-conotoxin-like augertoxins.
Keywords: Conoidea; Turridae; Polystira albida; Turritoxin; P-conotoxin; Framework IX;

This is the first report on the structural identity of a neuropeptide of the insect order Megaloptera. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca glands of the alderfly, Sialis lutaria. The sequence of the peptide was deduced from the multiple MSN electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Thr-Phe-Thr-Pro-Ser-Trp amide. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by comparing retention time on reversed-phase HPLC and establishing co-elution with the synthetic Leu2-form which also had exactly the same MS2 mass spectra as the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which has already more than 40 members. Interestingly, the primary structure is identical to that predicted from genome information for the adipokinetic hormone of the yellow fever mosquito, Aedes aegypti. Since alderflies are not known for their active flight metabolism but produce a rather high number of eggs, it is anticipated that the alderfly is a good study object to establish a possible role of the novel peptide to regulate fat mobilization from the fat body and transport into the egg, thereby playing a role in the control of reproductive processes.
Keywords: Arthropods; Insects; Megaloptera; Alderfly; Neuropeptide; Adipokinetic hormone family; Mass spectrometry;

Neuropeptides in Heteroptera: Identification of allatotropin-related peptide and tachykinin-related peptides using MALDI-TOF mass spectrometry by Susanne Neupert; William K. Russell; David H. Russell; Juan D. López; Reinhard Predel; Ronald J. Nachman (483-488).
Recently, the peptidomic analysis of neuropeptides from the retrocerebral complex and abdominal perisympathetic organs of polyphagous stinkbugs (Pentatomidae) revealed the group-specific sequences of pyrokinins, CAPA peptides (CAPA-periviscerokinins/PVKs and CAPA-pyrokinin), myosuppressin, corazonin, adipokinetic hormone, and short neuropeptide F. In this study, we used mass spectrometric profiling of nervous tissue from the species-rich taxon Hemiptera to identify products of two previously unobserved neuropeptide genes from these species, namely allatotropin-related peptide (ATRP) and tachykinin-related peptides (TKRPs). Since neither TKRPs nor allatotropin are accumulated in neurohemal organs, immunocytochemical data were analyzed to find potential accumulation sites within the central nervous system. By mass spectrometry, TKRPs were found to be accumulated in the antennal lobes, and ATRP was identified in the most posterior region of the abdominal ventral nerve cord and fourth abdominal nerves. In addition to neuropeptides from stink bugs, TKRPs and ATRP were also identified from the distantly related bugs Oncopeltus fasciatus (Lygaeidae) and Pyrrhocoris apterus (Pyrrhocoridae). In total, six TKRPs and one ATRP from each species could be elucidated by tandem mass spectrometry. The ATRP of all species is sequence-identical with Locusta migratoria accessory gland myotropin-1 (Lom-AG-MT-1), a member of the highly conserved insect allatotropin family.
Keywords: Tachykinin-related peptides; Allatotropin-related peptide; Mass spectrometry; Insect neuropeptides; Heteroptera; Nezara viridula; Acrosternum hilare; Banasa dimiata; Euschistus servus; Pentatoma rufipes; Oncopeltus fasciatus; Pyrrhocoris apterus;

The first insect allatotropin-related peptide (ATRP) was isolated from head extracts of the adult sphinx moth Manduca sexta [Kataoka H, Toschi A, Li JP, Carney RL, Schooley DA, Kramer SJ. Identification of an allatotropin from adult Manduca sexta. Science 1989;243:1481–3.]. Meanwhile ATRPs are known from different holometabolous insects but only a single ATRP could be identified from hemimetabolous insects [Paemen L, Tips A, Schoofs L, Proost P, Van Damme J, De Loof A. Lom-AG-myotropin: a novel myotropic peptide from the male accessory glands of Locusta migratoria. Peptides 1991;12:7–10.]. This means that the extensive analysis of neuropeptides from Leucophaea maderae and Periplaneta americana, which led to the discovery of many novel insect neuropeptides, did not result in the detection of any ATRP. In this study, we used another approach to find a cockroach ATRP by first identifying Manse-AT immunoreactive neurons in the terminal ganglion that can be stained by retrograde labeling and are suitable for dissection and subsequent mass spectrometric analysis. The peptidomic analysis of these putative ATRP neurons paved the way for the identification of the first cockroach ATRP. MALDI-TOF/TOF tandem mass spectrometry revealed a sequence identity with Locmi-AG-MT-1 which classifies this ATRP as a highly conserved neuropeptide. A mass spectrometric screening of the nervous system allowed the detection of ATRP-ion signals in different parts of the CNS of P. americana as well as L. maderae. The data obtained in this study will be incorporated in a map of peptidergic neurons from the CNS of the American cockroach, P. americana.
Keywords: Single cell peptidomics; Mass spectrometry; Allatotropin-related peptide; Lom-AG-myotropin I; Neuropeptide; Insect; Cockroach; Periplaneta americana;

The allatostatins (ASTs), with a Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide C-terminus, are neuropeptides that occur in many orders of insects, but are known to inhibit juvenile hormone (JH) synthesis by corpora allata (CA) only in cockroaches, crickets, and termites. 5 AST peptides with similar sequences to those of 6 species of cockroaches have been isolated and sequenced from extract of brain tissue of the termite Reticulitermes flavipes. The amino acid sequence of a 6th peptide, R. flavipes AST-7, determined by LC–MS/MS following HPLC fractionation of brain extract, is S-P-S-S-G-N-Q-R-L-Y-G-F-G-L-NH2. The 8 terminal amino acids are identical to AST-7 of the cockroach Diploptera punctata. R. flavipes and D. punctata AST-7s inhibited JH synthesis by CA of both species equally and their affinity for antibody against D. punctata AST-7 is similar. Immunoreactivity of termite tissue with this antibody indicates neuro- and myomodulatory activity of the peptide in addition to its demonstrated allatostatic function. The density of AST immunostaining in axons within the CA of R. flavipes and the rate of JH synthesis by similar glands were negatively correlated. This is evidence that when AST is abundant in the glands it is being released in vivo to limit JH production.
Keywords: Allatostatin; Immunoreactivity; Juvenile hormone synthesis; Reticulitermes flavipes; Diploptera punctata; Termite; Cockroach; Corpora allata;

Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.
Keywords: Bacterial expression system; Crustacean hyperglycemic hormone; Kuruma prawn; Marsupenaeus japonicus;

Neuropeptide-like precursor 4 is uniquely expressed during pupal diapause in the flesh fly by Aiqing Li; Joseph P. Rinehart; David. L. Denlinger (518-521).
Suppression subtractive hybridization comparing brains from diapausing and nondiapausing pupae of the flesh fly, Sarcophaga crassipalpis, suggested that the gene encoding neuropeptide-like precursor 4 (Nplp4) was uniquely expressed during diapause. We have sequenced the full-length cDNA encoding Nplp4 and used Northern blots to further evaluate linkage to diapause. The open reading frame of this cDNA encodes a 61-amino acid residue precursor protein containing a predicted 18 residue signal peptide, one 22-amino acid and one 2-amino acid propeptides, and a 19-amino acid neuropeptide. The amino acid sequence of the precursor protein shows 64% identity to Drosophila melanogaster Nplp4; homologues of this precursor protein are not known from species other than these two flies. Nplp4 mRNA levels were quite low in nondiapausing (long day) pupae, but in contrast the gene was highly upregulated in diapausing (short day) pupae. Expression increased at the onset of diapause, remained high throughout diapause, and then decreased 2 days after diapause was terminated. Although the function of this precursor protein and the neuropeptide it yields remain unknown, this close association with diapause suggests a potential role for Nplp4 in initiating and maintaining diapause in the flesh fly.
Keywords: Diapause; Overwintering; Neuropeptide-like precursor 4; Sarcophaga crassipalpis;

Cloning of neuropeptide-like precursor 1 in the gray flesh fly and peptide identification and expression by Peter Verleyen; Xi Chen; Szilvia Baron; Alice Preumont; Yue Jin Hua; Liliane Schoofs; Elke Clynen (522-530).
The neuropeptide-like precursor 1 (NPLP1) was first identified in a peptidomics experiment on Drosophila melanogaster. Limited data on this novel neuropeptide precursor suggest a role in the regulation of ecdysis in holometabolous larvae. In this study, we characterized the NPLP1 precursor in the gray flesh fly, Neobellieria bullata, which is an excellent model for physiological assays and hence to discover the role of the NPLP1 peptides. Antisera against three of the D. melanogaster NPLP1 neuropeptides stained an identical set of neurons in the central nervous system of N. bullata compared to D. melanogaster. A novel approach was applied to identify the N. bullata NPLP1 orthologs. Using a combination of affinity chromatography, mass spectrometry, cDNA cloning and RACE experiments, we obtained almost the complete coding sequence of the NPLP1 mRNA. Three encoded NPLP1 peptides were identified in central nervous system extracts by mass spectrometry. Neither doses of 25–250 pmol of synthetic Neb-MGYamide and Neb-PQNamide peptides, nor the NPLP1 antisera did affect the speed of retraction, contraction and tanning in the pupariation bioassay.
Keywords: Affinity chromatography; cDNA cloning; Immunocytochemistry; Neobellieria bullata; NPLP1; Neuropeptidomics;

GnRH in the brain and ovary of Sepia officinalis by Carlo Di Cristo; Emilia De Lisa; Anna Di Cosmo (531-537).
We have cloned from brain, ovary and eggs of the cephalopod Sepia officinalis a 269-bp PCR product, which shares 100% sequence identity with the open reading frame of GnRH isoform isolated from Octopus vulgaris. Similar to Octopus, this sequence encodes a peptide that is organized as a preprohormone from which, after enzymatic cleavage, a dodecapeptide is released. Apart from its length, this peptide shares all the common features of vertebrate GnRHs. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses followed by sequencing have confirmed that the same peptide transcript is also present in the ovary, as well as in eggs released in the mantle cavity. The use of an antibody made specifically against the oct-GnRH has revealed that the peptide is localized in the dorso-lateral basal and olfactory lobes, the two neuropeptidergic centers controlling the activity of the gonadotropic optic gland. Immunoreactive nerve endings are also present on the glandular cells of the optic glands. These results confirm the fact that, regardless of the evolutionary distances among animal phyla, GnRH is an ancient peptide present also in invertebrates, and also reinforce the notion that, despite the name “gonadotropin releasing-hormone” was attributed according to its role in vertebrates, probably this family of peptides always had a role in the broad context of animal reproduction. The divergence and spread of several different isoforms of this peptide among animals seem to be balanced, in both invertebrates and vertebrates, by the class-specificity of the GnRH isoform involved in reproductive processes.
Keywords: Cephalopods; Invertebrate neuroendocrinology; Reproduction; Cloning; Preprohormone;

Control of GnRH expression in the olfactory lobe of Octopus vulgaris by Carlo Di Cristo; Emilia De Lisa; Anna Di Cosmo (538-544).
In the cephalopod mollusk Octopus vulgaris, the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N-methyl-d-aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l-glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose–response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid (D-APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopus l-glutamate could be a key molecule in the nervous control of sexual maturation.
Keywords: Cephalopods; Invertebrate neuroendocrinology; Reproduction; Glutamate; Real-time PCR;

Characterization and distribution of NKD, a receptor for Drosophila tachykinin-related peptide 6 by Jeroen Poels; Ryan T. Birse; Ronald J. Nachman; Jakub Fichna; Anna Janecka; Jozef Vanden Broeck; Dick R. Nässel (545-556).
Neuropeptides related to vertebrate tachykinins have been identified in Drosophila and are referred to as drosotachykinins, or DTKs. Two Drosophila G protein-coupled receptors, designated NKD (neurokinin receptor from Drosophila; CG6515) and DTKR (Drosophila tachykinin receptor; CG7887), display sequence similarities to mammalian tachykinin receptors. Whereas DTKR was shown to be activated by DTKs [Birse RT, Johnson EC, Taghert PH, Nässel DR. Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides. J Neurobiol 2006;66:33–46; Poels J, Verlinden H, Fichna J, Van Loy T, Franssens V, Studzian K, et al. Functional comparison of two evolutionary conserved insect neurokinin-like receptors. Peptides 2007;28:103–8] and was localized by immunocytochemistry in Drosophila central nervous system (CNS), agonist-dependent activation and distribution of NKD have not yet been investigated in depth. In the present study, we have challenged NKD-expressing mammalian and insect cells with a library of Drosophila neuropeptides and discovered DTK-6 as a specific agonist that can induce a calcium response in these cells. In addition, we have produced antisera to sequences from NKD protein to analyze receptor distribution. We found that NKD is less abundantly distributed in the central nervous system than DTKR, and only NKD was found in the intestine. In fact, the two receptors are distributed in mutually exclusive patterns in the CNS. The combined distribution of the receptors in brain neuropils corresponds well with the distribution of DTKs. Most interestingly, NKD appears to be activated only by DTK-6, known to possess an Ala-substitution in an otherwise conserved C-terminal core motif. Our findings suggest that NKD and DTKR provide substrates for two functionally and spatially separated peptide signaling systems.
Keywords: Calcium; G protein-coupled receptor; Gut; insect; Neurokinin; Neuropeptide; Nervous system;

Solution conformations of an insect neuropeptide: Crustacean cardioactive peptide (CCAP) by Graham E. Jackson; Andre N. Mabula; Shane R. Stone; Gerd Gäde; Katalin E. Kövér; László Szilágyi; David van der Spoel (557-564).
The solution structure of crustacean cardioactive peptide (CCAP), a cyclic amidated nonapeptide neurohormone, was studied using molecular dynamics techniques, with constraints derived from NMR studies in water and water/dodecylphosphocholine micellar medium. This peptide, found in various invertebrates, has the primary sequence Pro1 Phe2 Cys3 Asn4 Ala5 Phe6 Thr7 Gly8 Cys9 NH2, with an intramolecular disulfide bridge between the two cysteine residues. In aqueous solution the peptide was found to have a type(IV) β-turn between residues 5–8. In a water/decane biphasic medium a type(IV) β-turn between residues 3 and 6 and two classic γ-turns between residues 4–6 and 7–9, were found. Analysis of the 1H and 13C NMR chemical shifts data showed that the model free S 2 order parameter of the residues varied between 0.65 and 0.9. The molecular dynamic root mean square fluctuations of structural ensembles of the backbone varied between 0.5 and 2.2 with the central residues showing the least fluctuations.
Keywords: CCAP; Arthropod neuropeptide; GROMACS; Molecular dynamics; Dodecylphosphocholine;

The degradation of 2 nmol synthetic leucomyosuppressin (LMS) by enzymes of the hemolymph, midgut lumen and midgut tissues of both Lacanobia oleracea and Spodoptera littoralis was investigated using reversed-phase high-performance liquid chromatography together with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Degradation of LMS in diluted hemolymph of L. oleracea and S. littoralis was not rapid, t 1/2  = 65.4 and 13.1 min, respectively, due to carboxypeptidase(s) and endopeptidase(s) present in the hemolymph. There was also some aminopeptidase activity in the hemolymph of both species. However, LMS was rapidly degraded by the diluted contents of the midgut lumen of L. oleracea and S. littoralis, t 1/2  = 0.5 and 2.2 min, respectively. The enzymes most likely responsible were trypsin-like serine protease(s) and carboxypeptidase(s). Degradation of LMS by midgut tissues containing 5 μg protein was not rapid in L. oleracea or S. littoralis, t 1/2  = 40.7 and 69.8 min, respectively. The most abundant degradation products probably resulted from carboxypeptidase activity, but some aminopeptidase activity was also detected.
Keywords: Neuropeptide; MALDI-TOF; Mass spectrometry; Insect;

Locomotor and geotactic behavior of Drosophila melanogaster over-expressing neprilysin 2 by Nicholas D. Bland; Philip Robinson; Josie E. Thomas; Alan D. Shirras; Anthony J. Turner; R. Elwyn Isaac (571-574).
The neprilysin (M13) family of zinc-metallopeptidases has been implicated in a variety of physiological processes, but principally the control of neuropeptide levels in a range of animal species. The over-expression of the amyloid-degrading enzyme, neprilysin, as a therapeutic strategy for Alzheimer's disease is a concept that is gaining in popularity. Here we utilize the GAL4/UAS system to over-express the Drosophila melanogaster Nep2 gene, a close homologue of neprilysin, in flies yielding an increase in NEP2 protein that is detectable by both immunoblotting and enzyme activity. This increase in NEP2 caused a behavioral phenotype manifested in abnormal climbing behavior. Wild type flies climb in a linear, vertical path, but NEP2 over-expressing (Nep2OEX) flies tend to climb in a spiral pattern and display an increase in grooming behavior during frequent stationary periods. Nep2OEX flies also perform poorly in a geotaxis maze, taking ten times as long to complete the course compared to wild type Drosophila. We hypothesize that the poor performance of the Nep2OEX flies in locomotor assays is due to perturbation of neuropeptide signaling and provides evidence of detrimental effects of neprilysin over-expression.
Keywords: Drosophila melanogaster; Neprilysin; Alzheimer's disease; Insect behavior; Neuropeptide metabolism;

Critical evaluation of the use of bioinformatics as a theoretical tool to find high-potential sources of ACE inhibitory peptides by Lieselot Vercruysse; Guy Smagghe; Arie van der Bent; Aart van Amerongen; Maté Ongenaert; John Van Camp (575-582).
A bioinformatics analysis to screen for high-potential sources of angiotensin converting enzyme (ACE) inhibitory peptides was conducted in the area of insect muscle proteins. Vertebrate muscle proteins are reported as good sources of ACE inhibitory peptides, while the research on invertebrate muscle proteins is limited. A phylogenetic tree constructed with actin sequences of both vertebrate and invertebrate species indicated a high homology. Furthermore, a quantitative in silico ACE inhibition analysis suggested that actin proteins of invertebrates have potentials as new sources of ACE inhibitory peptides. On one insect, Bombyx mori, a more detailed in silico analysis was done followed by a small experimental study. The in silico analysis indicated B. mori as a high-potential source of ACE inhibitory peptides and this was supported by the ACE inhibitory activity of the partially purified actin preparation. In conclusion, in food science, in silico analysis can be used as fast initial screening tool to look for high-potential sources of ACE inhibitory peptides and other peptidic bioactivities.
Keywords: Phylogeny; In silico ACE inhibition analysis; Actin; ACE inhibitory activity; Bombyx mori;

Cry1A toxins produced by Bacillus thuringiensis bind a cadherin receptor that mediates toxicity in different lepidopteran insect larvae. Insect cadherin receptors are modular proteins composed of three domains, the ectodomain formed by 9–12 cadherin repeats (CR), the transmembrane domain and the intracellular domain. Cry1A toxins interact with three regions of the Manduca sexta cadherin receptor that are located in CR7, CR11 and CR12 cadherin repeats. Binding of Cry1A toxin to cadherin induces oligomerization of the toxin, which is essential for membrane insertion. Also, it has been reported that cadherin fragments containing the CR12 region enhanced the insecticidal activity of Cry1Ab toxin to M. sexta and other lepidopteran larvae. Here we report that cadherin fragments corresponding to CR7 and CR11 regions also enhanced the activity of Cry1Ac and Cry1Ab toxin to M. sexta larvae, although not as efficient as the CR12 fragment. A single point mutation in the CR12 region (I1422R) affected Cry1Ac and Cry1Ab binding to the cadherin fragments and did not enhance the activity of Cry1Ab or Cry1Ac toxin in bioassays. Analysis of Cry1Ab in vitro oligomer formation in the presence of wild type and mutated cadherin fragments showed a correlation between enhancement of Cry1A toxin activity in bioassays and in vitro Cry1Ab-oligomer formation. Our data shows that formation of Cry1A toxin oligomer is in part responsible for the enhancement of Cry1A toxicity by cadherin fragments that is observed in vivo.
Keywords: Bacillus thuringiensis; Cry toxins; Toxin oligomerization; Cadherin receptor;

Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis by L. Pardo-López; C. Muñoz-Garay; H. Porta; C. Rodríguez-Almazán; M. Soberón; A. Bravo (589-595).
Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore-forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site-directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.
Keywords: Bacillus thuringiensis; Mode of action; Cry toxin; Pore toxin;

Conformational aspects and hyperpotent agonists of diapause hormone for termination of pupal diapause in the corn earworm by Qirui Zhang; Ronald J. Nachman; Pawel Zubrzak; David L. Denlinger (596-602).
Diapause hormone (DH) is a peptide well known to induce embryonic diapause in the commercial silkmoth Bombyx mori. More recently, this same neuropeptide was reported to break diapause in pupae of the agriculturally important Heliothis/Helicoverpa complex. In this study we examine the efficacy and potency of a select group of structural analogs of the native hormone in Helicoverpa zea and report the structures of several analogs that are considerably more potent than DH in breaking diapause. Among the most potent analogs (PK-Etz, PK-2Abf, 901) were those with structural components that enhance resistance to peptidases that degrade and inactivate the native peptide in vivo, which may account, at least in part, for the observed increase in potency for these analogs. Analog 901 was previously demonstrated to both enhance biostablility and bioavailability properties in adult heliothines and thus may be a potential candidate for topical application as a diapause-terminating agent. The significant activity observed for two restricted conformation analogs is consistent with an active conformation for diapause hormone that features a transPro within a type I β-turn in the C-terminal region. DH is also known to successfully break diapause only within a fairly narrow temperature range. While DH is effective at 21 °C, it is not effective at 18 °C. Likewise, the analogs were effective at 21 °C but not at 18 °C. By contrast, 20-hydroxyecdysone, a steroid hormone that is also capable of breaking diapause is effective at both temperatures, thus suggesting that DH and the ecdysteroids act through different mechanisms to terminate diapause.
Keywords: Diapause hormone; Diapause termination; Active core analogs; Aminopeptidases; Helicoverpa zea; Active conformation;

The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata by Charoula Kaskani; Constantine P. Poulos; JinRui Zhang; Stephen S. Tobe (603-607).
We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH46 and Dippu-DH31, on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH46, or slight inhibitory activity, in the case of DH31.
Keywords: Diuretic hormones; Diploptera punctata; Analogs; Methyl-homoserine replacement;

Biostable β-amino acid PK/PBAN analogs: Agonist and antagonist properties by Ronald J. Nachman; Orna Ben Aziz; Michael Davidovitch; Pawel Zubrzak; R. Elwyn Isaac; Allison Strey; Gloria Reyes-Rangel; Eusebio Juaristi; Howard J. Williams; Miriam Altstein (608-615).
The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a significant role in a multifunctional array of important physiological processes in insects. PK/PBAN analogs incorporating β-amino acids were synthesized and evaluated in a pheromonotropic assay in Heliothis peltigera, a melanotropic assay in Spodoptera littoralis, a pupariation assay in Neobellieria bullata, and a hindgut contractile assay in Leucophaea maderae. Two analogs (PK-βA-1 and PK-βA-4) demonstrate greatly enhanced resistance to the peptidases neprilysin and angiotensin converting enzyme that are shown to degrade the natural peptides. Despite the changes to the PK core, analog PK-βA-4 represents a biostable, non-selective agonist in all four bioassays, essentially matching the potency of a natural PK in pupariation assay. Analog PK-βA-2 is a potent agonist in the melanotropic assay, demonstrating full efficacy at 1 pmol. In some cases, the structural changes imparted to the analogs modify the physiological responses. Analog PK-βA-3 is a non-selective agonist in all four bioassays. The analog PK-βA-1 shows greater selectivity than parent PK peptides; it is virtually inactive in the pupariation assay and represents a biostable antagonist in the pheromonotropic and melanotropic assays, without the significant agonism of the parent hexapeptide. These analogs provide new, and in some cases, biostable tools to endocrinologists studying similarities and differences in the mechanisms of the variety of PK/PBAN mediated physiological processes. They also may provide leads in the development of PK/PBAN-based, insect-specific pest management agents.
Keywords: β-Amino acids; PBAN; Sex pheromone biosynthesis; Cuticular melanization; Pupariation; Hindgut contraction; Insect neuropeptide agonist/antagonist;

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect by Ronald J. Nachman; Peter E.A. Teal; Orna Ben Aziz; Michael Davidovitch; Pawel Zubrzak; Miriam Altstein (616-621).
A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25–30% (130–150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.
Keywords: Amphiphilic; PBAN; Sex pheromone biosynthesis; Cuticular melanization; Insect neuropeptide antagonist; Cuticle penetration; Noctuid moths;

Pacifastin-related peptides: Structural and functional characteristics of a family of serine peptidase inhibitors by Bert Breugelmans; Gert Simonet; Vincent van Hoef; Sofie Van Soest; Jozef Vanden Broeck (622-632).
Members of the pacifastin family are serine peptidase inhibitors, found in arthropods and have many members within different insect orders. Based on their structural characteristics, inhibitors of this peptide family are divided into two groups (I and II). Members of both groups exhibit specificity towards different types of serine peptidases. In addition, group I inhibitors display species selectivity. The specificity and selectivity of these inhibitors depends on the nature of their P1 residue and on additional interaction sites at the inhibitor's surface. Functional analysis studies have shown that crustacean pacifastin plays a key role in the immune response, whereas insect pacifastin-like peptides have multiple regulatory functions in processes involved in immunity, reproduction, phase transition, etc.
Keywords: Pacifastin-related peptide; Serine peptidase inhibitor; Arthropod; Insect; Immune response; Species selectivity;