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Ronald J. Nachman - One of the best experts on this subject based on the ideXlab platform.
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tick capa propeptide cdnas and receptor activity of endogenous tick Pyrokinins and analogs towards discovering Pyrokinin function in ticks
Peptides, 2021Co-Authors: Caixing Xiong, Ronald J. Nachman, Yunlong Yang, Patricia V PietrantonioAbstract:Abstract Pyrokinins (PKs) are pleiotropic neuropeptides with significant roles in invertebrate physiology. Although functions of PKs are known in insects, there is a lack of knowledge of PK-encoding genes and PKs functions in ticks. Herein the first tick cDNAs of the capability (capa) gene were cloned from the southern cattle tick, Rhipicephalus microplus (Acari: Ixodidae), and the blacklegged tick, Ixodes scapularis. Each cDNA encoded one periviscerokinin and five different Pyrokinins. Two PKs were identical in sequence in the two species. The three PKs unique to R. microplus (Rhimi-CAPA-PK1, -PK2, and -PK5) were tested on the recombinant R. microplus Pyrokinin receptor using a calcium bioluminescence assay. The Rhimi-CAPA-PKs acted as agonists with EC50s ranging from 101–188 nM. Twenty PK analogs designed for enhanced bioavailability and biostability were tested on the receptor. Five of these were designed based on the sequences of the three unique Rhimi-CAPA-PKs. Eight PK analogs were also agonists; four of them were full agonists that exhibited comparable efficacy to the native Rhimi-CAPA-PKs, with EC50 ranging from 401 nM-1.9 μM. The structure-activity relationships (SAR) of all analogs were analyzed. Our results suggested that a positively charged, basic lysine at the variable position X of the PK active core (FXPRLamide) conferred enhanced affinity to the analogs in their interaction with the tick receptor. These analogs are promising tools to elucidate the Pyrokinin function in ticks in vivo as these analogs are expected to have prolonged hemolymph residence time in comparison to the native peptides.
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solid phase synthesis of an insect Pyrokinin analog incorporating an imidazoline ring as isosteric replacement of a trans peptide bond
Molecules, 2021Co-Authors: Krzysztof Kaczmarek, Janusz Zabrocki, Barbara Pacholczyksienicka, łukasz Albrecht, Ronald J. NachmanAbstract:A facile solid-phase synthetic method for incorporating the imidazoline ring motif, a surrogate for a trans peptide bond, into bioactive peptides is reported. The example described is the synthesis of an imidazoline peptidomimetic analog of an insect Pyrokinin neuropeptide via a cyclization reaction of an iminium salt generated from the preceding amino acid and 2,4-diaminopropanoic acid (Dap).
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FXPRLamide (Pyrokinin/PBAN) Family
2016Co-Authors: Miriam Altstein, Aliza Hariton, Ronald J. NachmanAbstract:The Pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) family is one of the most studied neuropeptide (Np) families in insects and is one of the most important in insect physiol-ogy regulation. Physiological functions regulated by the PK/PBAN peptides include development, mating, muscle contraction, and tanning. Members of the family are found in many Lepidopteran and other insect species. In this chapter, we briefly present a histori-cal perspective of the discovery of the PK/PBAN peptides, provide details on the structure of the PK/PBAN genes and the processing of their prohormone to bioactive peptides, describe the distribution of the mRNA and peptides in the insect nervous system, and sum-marize the current knowledge on the PK/PBAN receptors, their signaling mechanisms, and their biological activity. Employment of the PK/PBAN family of peptides as a basis for designing a novel generation of insect-control agents based on Np antagonists is also briefly discussed
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Molecular and pharmacological characterization of the Chelicerata Pyrokinin receptor from the southern cattle tick, Rhipicephalus (Boophilus) microplus.
Insect biochemistry and molecular biology, 2015Co-Authors: Yunlong Yang, Ronald J. Nachman, Patricia V PietrantonioAbstract:Abstract We identified the first Pyrokinin receptor ( Rhimi - PKR ) in Chelicerata and analyzed structure-activity relationships of cognate ligand neuropeptides and their analogs. Based on comparative and phylogenetic analyses, this receptor, which we cloned from larvae of the cattle tick Rhipicephalus microplus (Acari: Ixodidae), is the ortholog of the insect Pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN)/diapause hormone (DH) neuropeptide family receptor. Rhimi -PKR functional analyses using calcium bioluminescence were performed with a developed stable recombinant CHO-K1 cell line. Rhimi -PKR was activated by four endogenous PKs from the Lyme disease vector, the tick Ixodes scapularis (EC 50 s range: 85.4 nM-546 nM), and weakly by another tick PRX-amide peptide, periviscerokinin (PVK) (EC 50 = 24.5 μM). PK analogs with substitutions of leucine, isoleucine or valine at the C-terminus for three tick PK peptides, Ixosc-PK1, Ixosc-PK2, and Ixosc-PK3, retained their potency on Rhimi -PKR. Therefore, Rhimi- PKR is less selective and substantially more tolerant than insect PK receptors of C-terminal substitutions of leucine to isoleucine or valine, a key structural feature that serves to distinguish insect PK from PVK/CAP 2b receptors. In females, ovary and synganglion had the highest Rhimi-PKR relative transcript abundance followed by the rectal sac, salivary glands, Malpighian tubules, and midgut. This is the first pharmacological analysis of a PK/PBAN/DH-like receptor from the Chelicerata, which will now permit the discovery of the endocrinological roles of this neuropeptide family in vectors of vertebrate pathogens.
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peptidomics applied a new strategy for development of selective antagonists agonists of insect Pyrokinin fxprlamide family using a novel conformational mimetic motif
Eupa Open Proteomics, 2014Co-Authors: Ronald J. NachmanAbstract:Abstract Applied peptidomics: A PK active core analog incorporating a novel transPro conformational-mimetic motif, the dihydroimidazole moiety, was found to demonstrate pure, selective agonism in Pyrokinin (PK) family bioassays. A second PK core analog incorporating the dihydroimidazole moiety proved to be an antagonist of the diapause-termination activity of another PK assay. The dihydroimidazole analogs feature a modification adjacent to the primary tissue-bound peptidase hydrolysis site that is expected to enhance biostability over natural PK peptides identified by peptidomics. The research identifies a novel scaffold to design mimetic PK analogs as potential environmentally favorable pest management agents capable of disrupting PK-regulated systems.
Manyeon Choi - One of the best experts on this subject based on the ideXlab platform.
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molecular and functional characterization of Pyrokinin like peptides in the western tarnished plant bug lygus hesperus hemiptera miridae
Insects, 2021Co-Authors: Joe J Hull, Manyeon Choi, Colin S Brent, Zsanett Miko, Jozsef Fodor, Adrien FonagyAbstract:The Pyrokinin (PK) family of insect neuropeptides, characterized by C termini consisting of either WFGPRLamide (i.e., PK1) or FXPRLamide (i.e., PK2), are encoded on the capa and pk genes. Although implicated in diverse biological functions, characterization of PKs in hemipteran pests has been largely limited to genomic, transcriptomic, and/or peptidomic datasets. The Lygus hesperus (western tarnished plant bug) PK transcript encodes a prepropeptide predicted to yield three PK2 FXPRLamide-like peptides with C-terminal sequences characterized by FQPRSamide (LyghePKa), FAPRLamide (LyghePKb), and a non-amidated YSPRF. The transcript is expressed throughout L. hesperus development with greatest abundance in adult heads. PRXamide-like immunoreactivity, which recognizes both pk- and capa-derived peptides, is localized to cells in the cerebral ganglia, gnathal ganglia/suboesophageal ganglion, thoracic ganglia, and abdominal ganglia. Immunoreactivity in the abdominal ganglia is largely consistent with capa-derived peptide expression, whereas the atypical fourth pair of immunoreactive cells may reflect pk-based expression. In vitro activation of a PK receptor heterologously expressed in cultured insect cells was only observed in response to LyghePKb, while no effects were observed with LyghePKa. Similarly, in vivo pheromonotropic effects were only observed following LyghePKb injections. Comparison of PK2 prepropeptides from multiple hemipterans suggests mirid-specific diversification of the pk gene.
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identification and characterization of gpcrs for Pyrokinin and capa peptides in the brown marmorated stink bug halyomorpha halys hemiptera pentatomidae
Frontiers in Physiology, 2020Co-Authors: Robert Vander K Meer, Seungjoon Ahn, Jacob Corcoran, Manyeon ChoiAbstract:The brown marmorated stink bug, Halyomorpha halys, is an invasive hemipteran that causes significant economic losses to various agricultural products around the world. Recently, the Pyrokinin and capa genes that express multiple neuropeptides were described in this species. Here we report six Pyrokinin and capa GPCRs including two splice variants, and evaluate their (a) ability to respond to neuropeptides in cell-based assays, and (b) expression levels by RT-PCR. Functional studies revealed that the H. halys Pyrokinin receptor-1 (HalhaPK-R1a & b) responded to the Pyrokinin 2 (PK2) type peptide. RT-PCR results revealed that these receptors had little or no expression in the tissues tested, including the whole body, central nervous system, midgut, Malpighian tubules, and reproductive organs of males and females. HalhaPK-R2 showed the strongest response to PK2 peptides and a moderate response to Pyrokinin 1 (PK1) type peptides (= DH, diapause hormone), and was expressed in all tissues tested. HalhaPK-R3a & b responded to both PK1 and PK2 peptides. Their gene expression was restricted mostly to the central nervous system and Malpighian tubules. All PK receptors were dominantly expressed in the fifth nymph. HalhaCAPA-R responded specifically to CAPA-PVK peptides (PVK1 and PVK2), and was highly expressed in the Malpighian tubules with low to moderate expression in other tissues, and life stages. Of the six GPCRs, HalhaPK-R3b showed the strongest response to PK1. Our experiments associated the following peptide ligands to the six GPCRs: HalhaPK-R1a & b and HalhaPK-R2 are activated by PK2 peptides, HalhaPK-R3a & b are activated by PK1 (= DH) peptides, and HalhaCAPA-R is activated by PVK peptides. These results pave the way for investigations into the biological functions of H. halys PK and CAPA peptides, and possible species-specific management of H. halys.
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identification and characterization of capa and Pyrokinin genes in the brown marmorated stink bug halyomorpha halys hemiptera gene structure immunocytochemistry and differential expression
Archives of Insect Biochemistry and Physiology, 2018Co-Authors: Seungjoon Ahn, Manyeon ChoiAbstract:CAPA and Pyrokinin (PK) neuropeptides are produced from two different genes, capa and Pyrokinin, respectively. In this study, we identified and characterized the capa and Pyrokinin genes from the brown marmorated stink bug, Halyomorpha halys (Hemiptera). The capa gene encodes two CAPA-PVK (periviscerokinin) peptides (DAGLFPFPRVamide and EQLIPFPRVamide) and one CAPA-DH (diapause hormone; NGASGNGGLWFGPRLamide). The Pyrokinin gene encodes three PK2 peptides (QLVSFRPRLamide, SPPFAPRLamide, and FYAPFSPRLamide). The whole-mounting immunocytochemistry revealed the neurons contained PRXamide-like peptides throughout the cerebral ganglia (CRG), gnathal ganglia (GNG), thoracic ganglia (TG), and abdominal ganglia (AG). A pair of neurosecretory cells in the CRG and three cell clusters in the GNG were found with the axonal projections extended through the lateral side. A pair of immunostained cells were found in the TG, while three pairs of cells were present in the fused AG. Different expression patterns of capa and Pyrokinin genes were observed in the CRG-GNG, TG, and AG. The capa gene was highly expressed in the AG tissue, whereas the Pyrokinin gene was strongly expressed in the CRG-GNG. Interestingly, different developmental stages showed similar expressions of both genes, with the highest from the first nymph, gradually decreasing to the female adult. Comparison of peptide sequences encoded from Pyrokinin genes showed the PK1 peptide is lost in Heteroptera suborders including H. halys, but retained in other suborders. The missing PK1 from the Pyrokinin gene might be compensated by CAPA-DH (=PK1-like) produced by the capa gene.
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identification and characterization of Pyrokinin and capa peptides and corresponding gpcrs from spotted wing drosophila drosophila suzukii
General and Comparative Endocrinology, 2017Co-Authors: Manyeon Choi, Seungjoon Ahn, Young A Kim, Young Ho KohAbstract:The family of FXPRLamide peptides serves as a major insect hormone. It is characterized by a core active amino acid sequence conserved at the C-terminal ends, and provides various physiological roles across the Insecta. In this study we identified and characterized Pyrokinin (PK) and CAPA cDNAs encoding two FXPRLamide peptides, Pyrokinin and CAPA-DH (diapause hormone), and two corresponding G protein-coupled receptors (GPCRs) from spotted wing drosophila (SWD), Drosophila suzukii. Expressions of PK and CAPA mRNAs were differentially observed during all life stages except the embryo, and the detection of CAPA transcription was relatively strong compared with the PK gene in SWD. Both D. suzukii Pyrokinin receptor (DrosuPKr) and CAPA-DH receptor (DrosuCAPA-DHr) were functionally expressed and confirmed through binding to PK and DH peptides. Differential expression of two GPCRs occurred during all life stages; a strong transcription of DrosuPKr was observed in the 3rd instar. DrosuCAPA-DHr was clearly expressed from the embryo to the larva, but not detected in the adult. Gene regulation during the life stages was not synchronized between ligand and receptor. For example, SWD CAPA mRNA has been up-regulated in the adult while CAPA-DHr was down-regulated. The difference could be from the CAPA mRNA translating multiple peptides including CAPA-DH and two CAPA-PVK (periviscerokinin) peptides to act on different receptors. Comparing the genes of SWD PK, CAPA, PKr and CAPA-DHr to four corresponding genes of D. melanogaster, SWD CAPA and the receptor are more similar to D. melanogaster than PK and the receptor. These data suggest that the CAPA gene could be evolutionally more conserved to have a common biological role in insects. In addition, the effect of Kozak sequences was investigated by the expression of the GPCRs with or without Kozak sequences in Sf9 insect cells. The Kozak sequenced PK receptor was significantly less active than the original (= no Kozak sequenced) receptor. Our results provide a knowledge for potential biological function(s) of PK and CAPA-DH peptides in SWD, and possibly offer a novel control method for this pest insect in the future.
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identification and expression of a new member of the Pyrokinin pban gene family in the sand fly phlebotomus papatasi
Journal of Insect Physiology, 2015Co-Authors: Manyeon Choi, Robert Vander K Meer, Neil D Sanscrainte, Alden S Estep, James J BecnelAbstract:The major family of neuropeptides (NPs) derived from the pk (Pyrokinin)/pban (pheromone biosynthesis activating neuropeptide) gene are defined by a common FXPRL-NH2 or similar sequence at the C-termini. This family of peptides has been found in all insect groups investigated to date and is implicated in regulating various physiological functions, including pheromone biosynthesis and diapause, but other functions are still largely unknown in specific life stages. Here we identify two isoforms of pk/pban cDNA encoding the PBAN domain from the sand fly Phlebotomus papatasi. The two pk/pban isoforms have the same sequence except for a 63 nucleotide difference between the long and short forms, and contain no alternative mRNA splicing site. Two NP homologues, DASGDNGSDSQRTRPPFAPRLamide and SLPFSPRLamide are expected, however, sequence corresponding to the diapause hormone was not found in the P. papatasi pk/pban gene. The PBAN-like amino acid sequence homologue SNKYMTPRL is conserved in the gene, but there is no cleavage site for processing a functional peptide. Characterizing the expression of the isoforms in developmental stages and adults indicates that the short form is differentially transcribed depending on the life stage. The P. papatasi pk/pban gene is the only known pk/pban gene with two transcriptional isoforms and from examination of endoproteolytic cleavage sites is expected to produce fewer peptides than most of the pk/pban genes elucidated to date; only Drosophila melanogaster is simpler with a single NP detected by mass spectroscopy. A phylogenetic analysis showed P. papatasi pk/pban grouped more closely with other nematoceran flies rather than higher flies.
Reinhard Predel - One of the best experts on this subject based on the ideXlab platform.
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serine phosphorylation of capa Pyrokinin in cockroaches a taxon specific posttranslational modification
Peptides, 2014Co-Authors: Sebastian Sturm, Reinhard PredelAbstract:In insects, posttranslational modifications of neuropeptides are largely restricted to C- and N-terminal amino acids. The most common modifications, N-terminal pyroglutamate formation and C-terminal α-amidation, may prevent a fast degradation of these messenger molecules. This is particularly important for peptide hormones. Other common posttranslational modifications of proteins such as glycosylation and phosphorylation seem to be very rare in insect neuropeptides. To check this assumption, we used a computer algorithm to search an extensive data set of MALDI-TOF mass spectra from cockroach tissues for ion signal patterns indicating peptide phosphorylation. The results verify that phosphorylation is indeed very rare. However, a candidate was found and experimentally verified as phosphorylated CAPA Pyrokinin (GGGGpSGETSGMWFGPRL-NH2) in the cockroach Lamproblatta albipalpus (Blattidae, Lamproblattinae). Tandem mass spectrometry revealed the phosphorylation site as Ser(5). Phosphorylated CAPA Pyrokinin was then also detected in most other cockroach lineages (e.g. Blaberidae, Polyphagidae) but not in closely related blattid species such as Periplaneta americana. This is remarkable since the sequence of CAPA Pyrokinin is identical in Lamproblatta and Periplaneta. A consensus sequence of CAPA Pyrokinins of cockroaches revealed a conserved motif that suggests phosphorylation by a Four-jointed/FAM20C related kinase.
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Identification and Expression of Capa Gene in the Fire Ant, Solenopsis invicta
2014Co-Authors: Manyeon Choi, Susanne Neupert, Robert Vander K Meer, Rene Köhler, Reinhard PredelAbstract:Recent genome analyses suggested the absence of a number of neuropeptide genes in ants. One of the apparently missing genes was the capa gene. Capa gene expression in insects is typically associated with the neuroendocrine system of abdominal ganglia; mature CAPA peptides are known to regulate diuresis and visceral muscle contraction. The apparent absence of the capa gene raised questions about possible compensation of these functions. In this study, we re-examined this controversial issue and searched for a potentially unrecognized capa gene in the fire ant, Solenopsis invicta. We employed a combination of data mining and a traditional PCR-based strategy using degenerate primers designed from conserved amino acid sequences of insect capa genes. Our findings demonstrate that ants possess and express a capa gene. As shown by MALDI-TOF mass spectrometry, processed products of the S. invicta capa gene include three CAPA periviscerokinins and low amounts of a Pyrokinin which does not have the C-terminal WFGPRLa motif typical of CAPA Pyrokinins in other insects. The capa gene was found with two alternative transcripts in the CNS. Within the ventral nerve cord, two capa neurons were immunostained in abdominal neuromeres 2–5, respectively, and projected into ventrally located abdominal perisympathetic organs (PSOs), which are the major hormone release sites of abdominal ganglia. The ventral location of these PSOs is a characteristic feature and was also found in another ant, Atta sexdens.
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List of peptides from capa and pk/pban genes of S. invicta, identified by MALDI-TOF/TOF mass spectrometry in this study.
2014Co-Authors: Manyeon Choi, Susanne Neupert, Robert Vander K Meer, Rene Köhler, Reinhard PredelAbstract:CPP, C-terminal precursor peptide; NPP, N-terminal precursor peptide; PBAN, pheromone biosynthesis activating neuropeptide; PVK, periviscerokinin; PK, Pyrokinin; aPSO, abdominal PSO; CC, corpora cardiaca.1mass match only.
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MALDI-TOF mass fingerprint spectrum (m/z 800–3000) from a preparation of corpora cardiaca from a female S. invicta (direct tissue profiling).
2014Co-Authors: Manyeon Choi, Susanne Neupert, Robert Vander K Meer, Rene Köhler, Reinhard PredelAbstract:Ion signals indicating the presence of products from the pk/pban gene are labeled; their sequences were subsequently confirmed by MS/MS experiments (not shown). The ion at m/z 2588.6 matched the predicted mass of CPP from the CAPA precursor but ion intensity of this peptide was not sufficient for MS/MS experiments. CPP, C-terminal precursor peptide (in the mass spectrum, CPP processed from the PK/PBAN precursor is designated); cor, corazonin; MS, myosuppressin (pQ/Q forms); PK, Pyrokinin.
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MALDI-TOF mass spectra from a preparation of a single abdominal PSO of female S. invicta (direct tissue profiling).
2014Co-Authors: Manyeon Choi, Susanne Neupert, Robert Vander K Meer, Rene Köhler, Reinhard PredelAbstract:A) Mass fingerprint spectrum (m/z 750–2700); prominent signals indicating the presence of three PVKs and the NPP (long transcript form) are detectable. The predicted PK shows a very weak signal intensity. This low signal intensity is likely a result of incomplete cleavage from the remaining C-terminus of the precursor sequence. With the exception of the supposed CPP at m/z 2588.5, sequences of all designated peptides were confirmed by MS/MS analyses. B) MALDI MS/MS fragment spectrum of the ion signal at m/z 974.6 (see 3A) under conditions of CID off. Fragment series (b- and y-type ions are labelled) confirmed the sequence of S. invicta PVK-2. aPSO, abdominal perisympathetic organ; PVK, periviscerokinin; PK, Pyrokinin; CPP, C-terminal precursor peptide; NPP, N-terminal precursor peptide.
Donghun Kim - One of the best experts on this subject based on the ideXlab platform.
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Functional Characterization of Ecdysis Triggering Hormone Receptors (AgETHR-A and AgETHR-B) in the African Malaria Mosquito, Anopheles gambiae
'Frontiers Media SA', 2021Co-Authors: Yoonseong Park, Vikas Jindal, Donghun KimAbstract:Insect ecdysis behavior, shedding off the old cuticle, is under the control of specific neuropeptides with the top command by the ecdysis triggering hormone (ETH). We characterized the ETH receptor (ETHR) of the malaria mosquito, Anopheles gambiae, by manual annotation of the NCBI gene (AGAP002881) and functional analysis, using a heterologous expression system in a mammalian cell line. The two splicing variants of ETHRs, ecdysis triggering hormone receptors (AgETHR-A and AgETHR-B), a conserved feature among insects, included of four (552 aa) and five exons (635 aa), respectively. The main feature of manual annotation of the receptor was a correction of N-terminal and exon-intron boundaries of an annotated gene (AGAP002881). Interestingly, the functional expression of the receptor in Chinese hamster ovary cells required modification of the transcription initiation site for mammalian Kozak consensus. In the calcium mobilization assay using the heterologous expression of each receptor, AgETHR-B showed a higher sensitivity to AgETH-1 (28 times) and AgETH-2 (320 times) than AgETHR-A. The AgETHRs showed specificity only to the ETH group of peptides but not to other groups carrying the C-termini motifs as PRXamide, such as Pyrokinin1/DH and Pyrokinin2/PBAN. Ecdysis triggering hormone receptors (AgETHR-B) responded to different ETH variants of other insect species more promiscuously than AgETHR-A
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functional characterization of ecdysis triggering hormone receptors agethr a and agethr b in the african malaria mosquito anopheles gambiae
Frontiers in Physiology, 2021Co-Authors: Yoonseong Park, Vikas Jindal, Donghun KimAbstract:Insect ecdysis behavior, shedding off the old cuticle, is under the control of specific neuropeptides with the top command by the ecdysis triggering hormone (ETH). We characterized the ETH receptor (ETHR) of the malaria mosquito, Anopheles gambiae, by manual annotation of the NCBI gene (AGAP002881) and functional analysis, using the heterologous expression system in a mammalian cell line. The two splicing variants of ETHRs, AgETHR-A and AgETHR-B, a conserved feature among insects, consisted of four (552 aa) and five exons (635 aa), respectively. The main feature of manual annotation of the receptor was a correction of N-terminal and exon-intron boundaries of an annotated gene (AGAP002881). Interestingly, the functional expression of the receptor in Chinese hamster ovarian (CHO) cells required modification of the transcription initiation site for mammalian Kozak consensus. In the calcium mobilization assay using the heterologous expression of each receptor, AgETHR-B showed a higher sensitivity to AgETH-1 (28 times) and AgETH-2 (320 times) than AgETHR-A. The AgETHRs showed specificity only to the ETH group of peptides but not to other groups carrying the C-termini motifs as PRXamide, such as Pyrokinin1/DH and Pyrokinin2/PBAN. AgETHR-B responded to different ETH variants of other insect species more promiscuously than AgETHR-A. The system we have established for the AgETHRs in this study will allow us to expand the scope into developing the AgETHRs as a new pesticidal target.
Jan Zdarek - One of the best experts on this subject based on the ideXlab platform.
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diapause hormone in the corn earworm helicoverpa zea optimum temperature for activity structure activity relationships and efficacy in accelerating flesh fly pupariation
Peptides, 2008Co-Authors: Qirui Zhang, Ronald J. Nachman, Jan Zdarek, David L DenlingerAbstract:Abstract 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.
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A Beta-Amino Acid Pyrokinin Analog Induces Irregular Pupariation Behavior in Larvae of the Flesh Fly Sarcophaga bullata
2008Co-Authors: Ronald J. Nachman, Pawel Zubrzak, Howard J. Williams, Allison Strey, Jan ZdarekAbstract:Abstract : The developmental process of pupariation is accelerated by members of the Pyrokinin class of neuropeptides in larvae of the flesh fly Sarcophaga bullata. A Pyrokinin analog (Ac-Y[beta3Phe]TPRLamide), in which a Phe residue is replaced with a beta-amino acid, accelerates pupariation in this fly at a potency (0.2 pmol/larva) that matches that of the native Pyrokinin factor. At higher concentrations, this beta-amino acid Pyrokinin analog induces irregular pupariation behavior patterns that are suggestive of neurotoxic properties. Biostable analogs based on this structure may in future provide analog leads with the potential to disrupt the important pupariation process in flies.)
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a comparison of the pupariation acceleration activity of Pyrokinin like peptides native to the flesh fly which peptide represents the primary pupariation factor
Peptides, 2006Co-Authors: Ronald J. Nachman, Pawel Zubrzak, Allison Strey, Jan ZdarekAbstract:Five native Pyrokinin-like peptides (Neb-PK-1, Neb-PK-2, Neb-PVK-1, [L 9 ]Neb-PVK-2, [I 9 ]NebPVK-2) identifiedin theneuropeptidomeofthefleshflyNeobellieria bullatawerecompared for their quantitative and/or qualitative effects on puparium formation (pupariation). In a standard pupariation bioassay, both Neb-PVK-1 and [I 9 ]Neb-PVK-2 proved inactive, whereas [L 9 ]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 NebPK-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 ffi 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 NebPK-2, rather than Neb-PK-1.
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Fraenkel's pupariation factor identified at last.
Developmental biology, 2004Co-Authors: Peter Verleyen, Jan Zdarek, Elke Clynen, Jurgen Huybrechts, Alfons Van Lommel, Luc Vanden Bosch, Arnold De Loof, Liliane SchoofsAbstract:Thirty-five years ago, Zdarek and Fraenkel demonstrated that nervous tissue extracts influenced development by accelerating pupariation in the grey flesh fly, Neobellieria bullata. We have now identified this pupariation factor as SVQFKPRLamide, designated Neb-Pyrokinin-2 (Neb-PK-2). To achieve this, the central nervous system of N. bullata wandering stage larvae, that is, preceding pupariation, were dissected and extracted before HPLC separation. Chromatographic fractions were screened with a bioassay for pupariation accelerating activity. Only one fraction showed huge pupariation activity. Mass spectrometry revealed the presence of a Pyrokinin, whose primary sequence could not be unequivocally determined by tandem mass spectrometry. However, this Neb-Pyrokinin appeared to be very prominent in the ring gland from which it was subsequently purified and identified. Synthetic Neb-PK-2 accelerates pupariation with a threshold dose of only 0.2 pmol and therefore, Neb-Pyrokinin is considered to be the genuine pupariation factor. The immunohistochemical distribution pattern of Neb-PK-2 is very similar to that of Drosophila Pyrokinin-2, from which it differs by only one amino acid residue. Hence, the recently identified G-protein coupled receptors (CG8784, CG8795) for Drosophila Pyrokinin-2 might play an important role in puparium formation.
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comparison of the effects of Pyrokinins and related peptides identified from arthropods on pupariation behaviour in flesh fly sarcophaga bullata larvae diptera sarcophagidae
Journal of Insect Physiology, 2004Co-Authors: Jan Zdarek, Peter Verleyen, Michael Mares, Lucie Doleckova, Ronald J. NachmanAbstract:Peptides from the Pyrokinin/PBAN family and some structurally related compounds identified in various arthropods were tested for acceleration of puparial contraction in flesh fly larvae. Modifications of behavioural patterns of pupariation were further studied for the active compounds using a behavioural analysis based on the recording of changes in tension of the cuticle. Nine peptides belonging to the Pyrokinin/PBAN family (Lem-PK, Pea-PK-5, Lom-PK II, Hez-PBAN, Bom-DH-I), identified in five different insect species, two Pyrokinin peptides derived from the genome of Drosophila melanogaster (capa-3, and hugin), and two Pyrokinins identified from the white shrimp Penaeus vannamei were very active in the pupariation assay, with threshold doses within the range of 0.1–5.0 pmol larva−1. High activity was also detected for a related peptide ETH1 from Drosophila. All of these peptides share a C-terminal PRLamide, which is essential and sufficient for the activity. Interestingly, two other structurally related peptides from Drosophila—ETH2 and capa-1—which feature conservative changes (Ile and Val, respectively) at the C-terminal Leu position, were inactive within a physiological range of concentrations. It is clear that the receptor mediating the acceleration of puparial contraction behaviour is sensitive to the introduction of greater steric bulk at the C-terminal Leu position. The peptides that accelerated pupariation showed very similar patterns of muscular and cuticular activity.
