Peptides (v.29, #2)
Announcement - Japan (V).
Special Issue Contents List (v-vi).
Editorial Board (CO2).
Introduction by Ronald J. Nachman (149-151).
Characterization of Aplysia Alb-1, a candidate water-borne protein pheromone released during egg laying by Scott F. Cummins; Bernard M. Degnan; Gregg T. Nagle (152-161).
Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-11–56 protein released during egg laying may serve a pheromonal function in concert with attractin, enticin, temptin and seductin.
Keywords: Aplysia; Alb-1; Attractin; Protein pheromone; Mollusk;
Comparative peptidomics of four related hemipteran species: Pyrokinins, myosuppressin, corazonin, adipokinetic hormone, sNPF, and periviscerokinins by Reinhard Predel; William K. Russell; David H. Russell; Juan Lopez; Jesus Esquivel; Ronald J. Nachman (162-167).
We performed the first comprehensive peptidomic analysis of neurohormones from hemipteran insects by analyzing the neuropeptides of two major neurohemal organs, namely the corpora cardiaca and abdominal perisympathetic organs. For the experiments we selected four related species of polyphagous stinkbugs (Pentatomidae), three of which are known to attack several important food crops. Peptide sequences were identified by MALDI-TOF mass spectrometry; tandem fragmentation of myosuppressin, sNPF, CAPA-periviscerokinins and pyrokinins revealed novel sequences not known from other insects so far. Most Leu/Ile and Glu/Lys ambiguities could be solved by either specific side-chain fragmentations or on-plate acetylation experiments. The identification of the specific sequences provides a solid basis for forthcoming pharmacological tests to study the neuroendocrine system of these pest insects. However, it should be mentioned in this context that the sequences of the peptides from different stinkbugs are likely not representative of Hemiptera in general. The forthcoming release of the genome from the reduviid Rhodnius prolixus will provide sufficient data to clear this point.
Keywords: FXPRLamide; CAPA-peptides; Mass spectrometry; Insect neuropeptides; Nezara viridula; Acrosternum hilare; Euschistus servus; Banasa dimiata; Pentatomidae;
Neuropeptides of the beetle, Tenebrio molitor identified using MALDI-TOF mass spectrometry and deduced sequences from the Tribolium castaneum genome by Robert J. Weaver; Neil Audsley (168-178).
Four neuropeptides were identified from the brain and corpora cardiaca-corpora allata (CC-CA) of the mealworm beetle Tenebrio molitor using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and information derived from the genome of the red flour beetle, Tribolium castaneum. Leucomyosuppressin (a FLRFamide), previously associated with cockroaches, but also subsequently identified from honey bee seen as a prominent peptide in both brain and CC-CA of T.molitor. A coding sequence for this peptide is found in the genome of T. castaneum. In addition, three FXPRLamides (pyrokinins), provisionally Tenmo-PK-1, Tenmo-PK-2 and Tenmo-PK-3 (HVVNFTPRLamide, SPPFAPRLamide, HL(I)SPFSPRLamide) were identified in both CC-CA and brain of T. molitor, again on the basis of predicted occurrence or similarity in T. castaneum. The sequence of Tenmo-PK-2 is the same as the PK-2 of the cockroach, Periplaneta americana. Other peptides readily predicted from the genome of T. castaneum include two AKH/HrTH peptides (Trica-AKH-1; pELNFSTDWamide and Trica-AKH-2; pELNFTPNWamide), the second of which is identical to Pyrap-AKH, an AKH-related peptide (Trica AKH-L; pEVTFSRDWPamide), two CRF-related diuretic factors (Trica-DH37 and Trica-DH47), the latter identical to Tenmo-DH47, a putative antidiuretic factor (Trica-ADFb; LYDDGSYKPHVYGF-OH), two sulfakinin-like peptides (Trica-SK-1; pETSDDY(SO3)GHLRFamide, and Trica SK-2; GEEPFDDYGHMRFamide), a potential allatostatin-C (Trica-AS; pESRYRQCYFNPISCF-OH), six allatostatin-B/myoinhibitory peptides (Trica-AST-B-1,2,3,4,5 & 6; DWNKDLHIWamide, GWNNLHEGWamide, AWQSLQSGWamide, NWGQFHGGWamide, SKWDNFRGSWamide, EPAWSNLGIWamide), an allatotropin-like peptide (Trica-ATL; GIEALKYHNMDLGTARGYamide), four ‘CAPA’-related peptides (Trica-CAPA-1,2,3,4; NKLASVYALTPSLRVamide, RIGKMVSFPRIamide, PGANSGGMWFGPRLamide, SENFTPWAYIILNGEAPIIREVHYSPRLamide), proctolin (RYLPT), a potential SIFamide (Trica-SIFa; TYRKPPFNGSIFamide), an arginine-vasopressin-related peptide (Trica-AVP; CLITNCPRGamide) and an ITP-related peptide (Trica-ITP). No evidence was found for the presence of ‘A’ allatostatins (Y/FxFGLamides) or corazonin, either in T. molitor, or in the genome of T. castaneum.
Keywords: Peptidomics; Insect; Coleoptera; Allatostatin; Leucomyosuppressin; Pyrokinin; Allatotropin;
Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice by Alejandro Zugasti-Cruz; Manuel B. Aguilar; Andrés Falcón; Baldomero M. Olivera; Edgar P. Heimer de la Cotera (179-185).
As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92 Da; calculated monoisotopic mass 2884.20 Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63 Da; calculated monoisotopic mass 3308.34 Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.
Keywords: Conoidea; Conidae; Cone snail; Conus austini; Conotoxins; Vermivorous; Worm-hunting; 4-Cys; Framework 14;
Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks by Manuel B. Aguilar; Karen S. Luna-Ramírez; Daniel Echeverría; Andrés Falcón; Baldomero M. Olivera; Edgar P. Heimer de la Cotera; María Maillo (186-195).
A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPMγDPLγIIRI-nh2, (γ, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72 Da; experimental monoisotopic mass, 1468.70 Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.
Keywords: Conoidea; Cone snail; Conus spurius; Conorfamide; Gamma-carboxyglutamate; FaRP;
Diapause hormone in the corn earworm, Helicoverpa zea: Optimum temperature for activity, structure–activity relationships, and efficacy in accelerating flesh fly pupariation by Qirui Zhang; Jan Zdarek; Ronald J. Nachman; David L. Denlinger (196-205).
Diapause hormone (DH) effectively terminated pupal diapause in Helicoverpa zea. This effect was temperature-dependent, with an optimum of 21 °C. The dose–response curve indicated an ED50 of DH for diapause termination of approximately 100 pmol. The core sequence and essential amino acids were determined by bioassays using modified and truncated DH analogs. A C-terminal hepta-peptide, LWFGPRLa, was the core sequence required for diapause termination. Activity was lost when Alanine was substituted for any of the amino acids in the hepta-peptide, with the exception of Glycine. A fragment series of analogs suggested that the amide and Arginine were the most important components needed for terminating diapause. Leucine, Tryptophan, and Phenylalanine at the N-terminus of the hepta-peptide were also critical for activity. The C-terminal Leucine was less important: deletion resulted in decreased activity, although it could not be substituted by Alanine. The fact that a portion of the DH sequence is similar to the pyrokinin that accelerates fly pupariation prompted us to also evaluate the capability of DH to accelerate development in the flesh fly, Sarcophaga bullata. The threshold dose of DH essential to accelerate fly pupariation was 5 pmol for immobilization/retraction and longitudinal contraction and 10 pmol for tanning, approximately one or two orders of magnitude lower than the effective dose required for diapause termination in H. zea. Tensiometric measurements revealed that DH affected neuromuscular patterns of pupariation behavior and associated cuticular changes in a manner similar to that of the fly pyrokinins and their analogs.
Keywords: Diapause hormone; Neuropeptide analogs; Diapause termination; Pupariation; Helicoverpa zea; Sarcophaga bullata;
Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus by V.A. Te Brugge; I. Orchard (206-213).
The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.
Keywords: Oncopeltus fasciatus; DH31; Peptide;
Peptidergic control of the heart of the stick insect, Baculum extradentatum by Aiza Ejaz; Angela B. Lange (214-225).
The dorsal vessel of the Vietnamese stick insect, Baculum extradentatum, consists of a tubular heart and an aorta that extends anteriorly into the head. Alary muscles, associated with the heart, are anchored to the body wall with attachments to the dorsal diaphragm. Alary muscle contraction draws haemolymph into the heart through incurrent ostia. Excurrent ostia lie on the dorsal vessel in the last thoracic and in each of the first two abdominal segments. Muscle fibers are associated with these excurrent ostia. Crustacean cardioactive peptide (CCAP)- and proctolin-like immunoreactivity is present in axons of the segmental nerves that project to the dorsal vessel, and in processes extending over the heart and alary muscles. Proctolin-like immunoreactive processes are also localized to the valves of the incurrent ostia and to the excurrent ostia. Neither the link nerve neurons, nor the lateral cardiac neurons, stain positively for these peptides. Physiological assays reveal dose-dependent increases in heart beat frequency in response to CCAP and proctolin. Isolating the dorsal vessel from the ventral nerve cord led to a change in the pattern of heart contractions, from a tonic, stable heart beat, to one which was phasic. The tonic nature was restored by the application of CCAP.
Keywords: Crustacean cardioactive peptide; Proctolin; Immunohistochemistry; Alary muscle; Excurrent ostia; Physiology;
Carbohydrate and lipid metabolism in cockroach (Periplaneta americana) fat body are both activated by low and similar concentrations of Peram-AKH II by Julia Michitsch; John E. Steele (226-234).
Injection of 0.1 pmol of the octapeptide Peram-AKH II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH2) elicits a significant hypertrehalosemic response in the American cockroach, Periplaneta americana; a maximal effect is obtained with 1 pmol. The latter amount also lowers the level of neutral lipid (NL) and phospholipid (PL) in the hemolymph. The evidence supports the idea that Peram-AKH II promotes the liberation of fatty acids from hemolymph phospholipid, and indirectly diacylglycerol in the same compartment. The fatty acids are then transported into the fat body where they are converted into triacylglycerol for storge. Because lipolysis and trehalose synthesis are initiated by a common concentration of Peram-AKH II it is reasonable to suggest that the physiological function of Peram-AKH II involves the participation of both metabolic pathways.
Keywords: Cockroach; Fat body; Fatty acids; Hypertrehalosemic hormone; Neutral lipid; Peram-AKH II; Phospholipid; Trehalose; Triacylglycerol synthesis;
The role of hemocytes, serine protease inhibitors and pathogen-associated patterns in prophenoloxidase activation in the desert locust, Schistocerca gregaria by Vanessa Franssens; Gert Simonet; Bert Breugelmans; Sofie Van Soest; Vincent Van Hoef; Jozef Vanden Broeck (235-241).
The prophenoloxidase-activating system is an important component of the innate immune response of insects, involved in wound healing and melanotic encapsulation. In this paper we show that in the desert locust, Schistocerca gregaria, hemocytes, challenged with microbial elicitors, are indispensable for the limited proteolytic activation of prophenoloxidase (proPO) in plasma. In addition, we assessed the influence of serine protease inhibitors on the induction of PO-activity in plasma. While soybean Bowman–Birk inhibitor (SBBI) inhibited the PO activation by laminarin-treated hemocytes, the endogenous pacifastin-related inhibitors, SGPI-1 (S. gregaria pacifastin-related inhibitor-1) and SGPI-2 did not affect the PO-activity under similar conditions. On the other hand, real-time PCR analysis revealed that the transcripts, encoding SGPI-1–3, were more abundant in the fat body of immune challenged animals, as compared to control animals.
Keywords: Innate immunity; Phenoloxidase; Peptide; Serine protease inhibitor; Insect; Hemolymph;
A putative farnesoic acid O-methyltransferase (FAMeT) orthologue in Drosophila melanogaster (CG10527): Relationship to juvenile hormone biosynthesis? by S.M. Burtenshaw; P.P. Su; J.R. Zhang; S.S. Tobe; L. Dayton; W.G. Bendena (242-251).
Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. Farnesoic acid O-methyltransferase (FAMeT) is thought to be an important enzyme in the JH biosynthetic pathway, catalyzing methylation of farnesoic acid (FA) to methyl farnesoate (MF). Previous evidence in other insects suggested that FAMeT is rate limiting and regulated by a neuropeptide family, the allatostatins. A full-length cDNA encoding a 296 amino acid putative FAMeT has been isolated. A recombinant (r)FAMeT was cloned, expressed and a specific antiserum generated. rFAMeT was assayed for enzymatic activity using a radiochemical assay. In this assay, no activity was detected either with rFAMeT alone or when added to a corpus allatum CA extract. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of Drosophila melanogaster ring gland. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on sesquiterpenoid biosynthesis. Drome AST appears to have a select effect on JH bisepoxide biosynthesis and not MF or JHIII. Additional analysis of MF, JHIII and JHB3 release in strains with a deficiency or decrease of FAMeT compared to wild type shows no significant decrease in MF, JHIII or JH bisepoxide synthesis. Deficiency strains that reduce the level of FAMeT showed reduced longevity relative to wildtype but this result may be due to other genetic influences.
Keywords: Juvenile hormone regulation; Allatostatin; Insect; Metamorphosis;
Characterization of the putative farnesoic acid O-methyltransferase (LvFAMeT) cDNA from white shrimp, Litopenaeus vannamei: Evidence for its role in molting by Jerome Ho Lam Hui; Stephen S. Tobe; Siu-Ming Chan (252-260).
Methyl farnesoate (MF) is the crustacean homolog of the insect juvenile hormone and is believed to regulate growth and reproduction in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to MF. Here we report the cloning and characterization of two forms of FAMeTs (i.e. LvFAMeT-S and LvFAMeT-L) from the shrimp Litopenaeus vannamei. LvFAMeT transcript has a wide tissue distribution pattern in L. vannamei and is also expressed in nauplius, zoea, mysis, post-larval stages and adults. Unlike FAMeTs reported in other decapods, transcripts of two different sizes were detected in L. vannamei. We postulate that the wide distribution of LvFAMeT expression may be related to its role in growth and regulation of molting. To study the functions of LvFAMeT in molting, the RNA interference (RNAi) technique was used. Injection of double stranded RNA (dsRNA) for LvFAMeT knocked down the expression of LvFAMeT in shrimp for at least 3 days and the shrimp did not advance to the final stage of molt cycle. Furthermore, the expression of the molt-related genes encoding cathepsin-L and the hemocyanin gene was disturbed. Subsequently, 100% mortality of the shrimp was observed in the LvFAMeT dsRNA-injected shrimp. In contrast, control shrimp completed their molt and proceeded to the next molt cycle. We postulate that, as an important enzyme for the conversion of FA to MF, RNAi injection knocked down the expression of LvFAMeT which could potentially result in a decrease in the production of MF and subsequently, could affect the molting process. The newly identified LvFAMeT may be involved in the control of molting in shrimp. The results of this study demonstrate the potential use of the RNA interference technique to study other putative genes identified in crustaceans.
Keywords: Farnesoic acid; Methyl farnesoate; Shrimp; Methyltransferase; Molting; In vivo; RNA interference;
Antihypertensive mechanism of the dipeptide Val-Tyr in rat aorta by Lieselot Vercruysse; Nicole Morel; John Van Camp; Justyna Szust; Guy Smagghe (261-267).
Antihypertensive peptides received much interest over the last decade. These peptides are known to be angiotensin converting enzyme (ACE) inhibitors in vitro, but the actual antihypertensive mechanisms in vivo are still unclear. In this research, we used rat aortic rings in organ bath experiments to investigate five potential vascular antihypertensive mechanisms of the dipeptide Val-Tyr. Only one significant effect was observed, namely preincubation of the aorta with Val-Tyr led to a significant shift of the concentration–response curve evoked by angiotensin I (Ang I). Val-Tyr had no effect on the angiotensin II receptor or the α-adrenergic receptor. Furthermore, it did not interact with voltage-operated Ca2+ channels, or with nitric oxide production/availability. In conclusion, our results show that Val-Tyr specifically inhibits Ang I-evoked contraction through ACE inhibition and that four other main mechanisms of vascular tone regulation are not affected.
Keywords: Organ bath; Rat aorta; Val-Tyr; Antihypertensive mechanism;
The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: Identification, functional expression, and structure–activity relationships of ligand analogs by Young-Joon Kim; Ronald J. Nachman; Karlygash Aimanova; Sarjeet Gill; Michael E. Adams (268-275).
Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure–activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3′-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X = T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5 > L6 > F2 ≫ P4 > T3 ≫ Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.
Keywords: Heliothis virescens; PBAN receptor; cDNA; Binding site peptidomimetic;
Molecular cloning and characterization of an allatostatin-like receptor in the cockroach Diploptera punctata by Panida Lungchukiet; B. Cameron Donly; Jinrui Zhang; Stephen S. Tobe; William G. Bendena (276-285).
Two Drosophila receptors (AlstR/DAR-1 and DAR-2) with sequence similarity to mammalian galanin receptors have been previously identified. These receptors have been shown to form specific interactions with neuropeptides that resemble cockroach allatostatins (ASTs), which have a characteristic Tyr/Phe-Xaa-Phe-Gly-Leu-NH2 carboxyl-terminus. We hypothesized that similar allatostatin receptors exist in the cockroach Diploptera punctata that may regulate the numerous effects that this family of peptides exerts on a range of target tissues. The polymerase chain reaction (PCR) was used, with primer design based on the Drosophila allatostatin receptor (AlstR). Using these primers, a putative allatostatin-like receptor cDNA was isolated from a λZAP-cDNA library prepared from the corpora allata of the D. punctata. As an approach to testing the function of this receptor in vivo, the technique of double-stranded RNA (dsRNA) gene interference was tested. Initial experiments suggest that the putative inhibition of receptor RNA expression may increase juvenile hormone (JH) production.
Keywords: Pest control; Cloning; Insect; G-protein coupled receptor; Real-time PCR; RNA interference; Juvenile hormone;
Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro by Neil Audsley; June Matthews; Ronald J. Nachman; Robert J. Weaver (286-294).
The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L–H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm2/h) measured after a 60-min incubation with 100 μmol/l of peptide in the lumen bathing fluid. The rates of flux, L–H and H–L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.
Keywords: Neuropeptide; MALDI-TOF; Mass spectrometry; Insect; Ussing chamber; ELISA;
Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus by S. Taneja-Bageshwar; A. Strey; K. Kaczmarek; J. Zabrocki; P.V. Pietrantonio; R.J. Nachman (295-301).
The multifunctional ‘insect kinins’ share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala; and are associated with the regulation of diuresis in a variety of species of insects. We previously reported the functional expression of a southern cattle tick (Boophilus microplus) G protein-coupled receptor that is activated by insect kinins. Four different stereochemical variants of each of the 4-aminopyroglutamic acid (APy) and tetrazole moieties, mimics of a cis-peptide bond, type VI β-turn in insect kinins were now evaluated on the expressed tick receptor using a calcium bioluminescence plate assay. This study represents the first investigation of the interaction of restricted-conformation analogs incorporating components that mimic specific conformations and/or peptide bond orientations in an expressed arthropod neuropeptide receptor. Analog Ac-RF[APy]WGa (2R,4S) was at least 10-fold more active than the other analogs, thus identifying the optimal stereochemistry for tick receptor interaction. The optimal stereochemistry for the tetrazole insect kinin analogs in the tick receptor assay was identified as (d,l). The APy is superior to the tetrazole as a scaffold for the design of mimetic insect kinin analogs. These biostable analogs provide new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin regulated processes.
Keywords: Kinin; Stereochemistry; Arthropod;
Identification of selective and non-selective, biostable β-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti by Suparna Taneja-Bageshwar; Allison Strey; Pawel Zubrzak; Howard Williams; Gloria Reyes-Rangel; Eusebio Juaristi; Patricia Pietrantonio; Ronald J. Nachman (302-309).
The multifunctional arthropod ‘insect kinins’ share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Eight different analogs of the insect kinin C-terminal pentapeptide active core in which the critical residues Phe1, Pro3 and Trp4 are replaced with β3-amino acid and/or their β2-amino acid counterparts were evaluated on recombinant insect kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini) and the dengue vector, the mosquito Aedes aegypti (L.). A number of these analogs previously demonstrated enhanced resistance to degradation by peptidases. Single-replacement analog β2Trp4 and double-replacement analog [β3Phe2, β3Pro3] of the insect kinins proved to be selective agonists for the tick receptor, whereas single-replacement analog β3Pro3 and double-replacement analog [β3Phe, β3Pro3] were strong agonists on both mosquito and tick receptors. These biostable analogs represent new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin-regulated processes.
Keywords: β-Amino acids; Kinins; Kinin receptors; Neuropeptides; Peptides;
FMRFamide and related peptides in the phylum mollusca by Estuardo López-Vera; Manuel B. Aguilar; Edgar P. Heimer de la Cotera (310-317).
FMRFamide is one of the well-known peptides studied within the phylum Mollusca. It was first isolated from the clam Macrocallista nimbosa during the end of the 1960s. Since then, a number of reports related to FMRFamide have been published from different experimental approaches, revealing that it and its related peptides (FaRPs) are implicated in a variety of physiological processes. As this year is the 30th anniversary since its discovery, this review focuses on diverse findings related to both FMRFamide and FaRPs in the phylum Mollusca.
Keywords: FMRFamide peptide; FaRPs; Mollusk;
The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta by N. Jiménez-Juárez; C. Muñoz-Garay; I. Gómez; S.S. Gill; M. Soberón; A. Bravo (318-323).
The insecticidal Cry toxins from Bacillus thuringiensis bacteria are pore-forming toxins that lyse midgut epithelial cells in insects. We have previously proposed that they form pre-pore oligomeric intermediates before membrane insertion. For formation of these oligomers coiled-coil structures are important, and helix α-3 from Cry toxins could form coiled-coils. Our data shows that different mutations in helix α-3 are affected in pore formation and toxicity. Mutants affected in toxicity bind Bt-R1 receptor with a similar K D as the wild type toxin but do not form oligomers nor induce pore formation in planar lipid bilayers, indicating that the pre-pore oligomer is an obligate intermediate in the intoxication of Cry1Ab toxin and that interaction of monomeric Cry1Ab with Bt-R1 is not enough to kill susceptible larvae.
Keywords: Bacillus thuringiensis; Cry toxins; Oligomerization; Pore-formation;
Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins by Luisa Elena Fernández; Isabel Gómez; Sabino Pacheco; Iván Arenas; Sarjeet S. Gilla; Alejandra Bravo; Mario Soberón (324-329).
Phage display is an in vitro method for selecting polypeptides with desired properties from a large collection of variants. The insecticidal Cry toxins produced by Bacillus thuringiensis are highly specific to different insects. Various proteins such as cadherin, aminopeptidase-N (APN) and alkaline phosphatase (ALP) have been characterized as potential Cry-receptors. We used phage display to characterize the Cry toxin–receptor interaction(s). By employing phage-libraries that display single-chain antibodies (scFv) from humans or from immunized rabbits with Cry1Ab toxin or random 12-residues peptides, we have identified the epitopes that mediate binding of lepidopteran Cry1Ab toxin with cadherin and APN receptors from Manduca sexta and the interaction of dipteran Cry11Aa toxin with the ALP receptor from Aedes aegypti. Finally we displayed in phages the Cry1Ac toxin and discuss the potential for selecting Cry variants with improved toxicity or different specificity.
Keywords: Phage display; Bacillus thuringiensis; Cry toxin;