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A De Loof

Publications and source records attributed to A De Loof.

At least 37 records · Page 2Linked to original sources
PubMed2002

New insights in Adipokinetic Hormone (AKH) precursor processing in Locusta migratoria obtained by capillary liquid chromatography-tandem mass spectrometry.

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

Animals↗
PubMed2002

Search for peptidic molecular markers in hemolymph of crowd-(gregarious) and isolated-reared (solitary) desert locusts, Schistocerca gregaria.

An HPLC analysis of hemolymph extracts was undertaken to uncover differences between desert locusts, Schistocerca gregaria, reared under either crowded or isolated conditions. Some differences in the chromatographic pattern could be detected. One of the major peaks in the hemolymph of crowd-reared adults was found to be a minor one in isolated-reared individuals, whereas other peaks increased after solitarization. The differences became even more pronounced after several generations of isolated rearing. The dominant chromatographic peak in hemolymph extracts of the crowd-reared animals was identified as a novel peptide with a molecular mass of 6080Da. Edman degradation in combination with enzymatic fragmentation and quadrupole-time of flight (Q-Tof) mass spectrometry revealed the full sequence: DNADEDTICVAADNKFYLYANSLKLYTCYNQLPKVYVVKPKSQCRSSLSDCPTS. This 54 aa-peptide is very abundant in hemolymph of crowd-reared adults. Its concentration in hemolymph amounts to 0.1mM. To uncover the function, its effects were investigated in several bioassays, so far without positive results. One of the other peaks differentially expressed in the individuals of the two phases was identified as SGPI-2 (MW=3794Da), which is a serine protease inhibitor in locusts.

Amino Acid Sequence↗
PubMed2002

cDNA cloning of two different serine protease inhibitor precursors in the migratory locust, Locusta migratoria.

Recently, a novel serine protease-inhibiting peptide family, designated as the 'pacifastin family', has been described in locusts and crayfish. All members of this family possess a characteristic cysteine-rich domain. The present study describes the cDNA cloning, sequencing and transcript distribution of two novel pacifastin-related peptide precursors in the migratory locust, Locusta migratoria. Only one of the encoded peptides (HI) was identified previously, whereas six others represent new members of the pacifastin family. Northern blot analysis showed that both precursor transcripts are present in adult locust fat body. These could not be detected in the midgut. Interestingly, an in silico data mining approach of the expressed sequence tags (EST) database revealed the existence of Manduca sexta and Bombyx mori cDNAs that display pronounced sequence similarities with these locust pacifastin-related transcripts.

Amino Acid Sequence↗
PubMed2002

Cloning of two cDNAs encoding isoforms of a pacifastin-related precursor polypeptide in the desert locust, Schistocerca gregaria: analysis of stage- and tissue-dependent expression.

A novel serine protease inhibitor peptide family, designated as the 'pacifastin family', has recently been described in insects (locusts, lepidopterans) and crustaceans (crayfish). This study presents the cDNA cloning of two isoforms of SGPP-3, a novel pacifastin-related precursor in the desert locust, Schistocerca gregaria, which codes for three putative inhibitor peptides. The precursor isoforms differ at a single amino acid position in the third, C-terminal peptide. Northern blot analysis confirmed the presence of two different transcripts (0.75 and 0.90 kb). Both transcripts are most abundant in the fat body and appear to be strongly regulated during the moulting cycle. In addition, the amount of transcript proved to be strictly regulated in the ovaries during the female reproductive cycle.

Amino Acid Motifs↗
PubMed2001

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells.

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.

Adenosine Monophosphate↗
PubMed2001

Gonadotropins in insects: an overview.

Control of gonad development in insects requires juvenile hormone, ecdysteroids, and a peptidic brain gonadotropin(s). Compared to vertebrates, the situation in insects with respect to the molecular structure of gonadotropins is far less uniform. Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) of vertebrates are glycoproteins that are synthezised in the hypothalamus and released from the anterior pituitary. They stimulate gonad development, the production of progesterone or of sex steroids (estrogens, androgens). None of the known insect gonadotropins is a glycoprotein, neither can they be grouped into a single peptide family. In Drosophila, two G-protein coupled receptors, structurally related to the mammalian glycoprotein hormone receptors, have been identified. Nothing is known about their natural ligands. The sex-steroids of insects are likely to be ecdysteroids (20E in females, E in males of some species). Some of the identified gonadotropins speed up vitellogenesis (locust OMP and some -PF/-RFamide peptides) or stimulate ecdysteroid production by the ovaries (locust-OMP and Aedes- OEH) or testis (testis ecdysiotropin of Lymantria). In flies, the only as yet identified gonadotropin is the cAMP-generating peptide of Neobellieria. The seeming absence of uniformity in gonadotropins in insects might be due to a multitude of factors that can stimulate ecdysteroid production and/or to the use of different bioassays. Arch.

Amino Acid Sequence↗
PubMed2001

Mass spectrometric evidence for the deficiency in the dark-color-inducing hormone,.

A factor present in the brain and corpus cardiacum responsible for the induction of dark colour in Locusta migratoria was recently isolated and identified from the corpora cardiaca of normally pigmented locusts. The purification of this factor, designated as [His7]-corazonin was monitored using an albino mutant from a laboratory colony of an Okinawa (Japan) strain. In this study, we provide unequivocal mass spectrometric evidence that the brain and the corpora cardiaca of this albino Locusta mutant are deficient in [His7]-corazonin. Previously, [His7]-corazonin was shown to be responsible for the induction of dark body colour patterns as observed in crowded locusts. Using nanoflow-liquid chromatography-mass spectrometry, we demonstrated that this dark colour-inducing hormone is, however, present in the corpora cardiaca of solitary locusts (Schistocerca gregaria). Arch.

Animals↗
PubMed2001

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

Testis ecdysiotropin, an insect gonadotropin that induces synthesis of ecdysteroid.

Testes of lepidoptera synthesized ecdysteroid in a somewhat different temporal pattern than the prothoracic glands that release ecdysteroid to the hemolymph. Brain extracts from Heliothis virescens and Lymantria dispar induced testes to synthesize ecdysteroid, but did not affect prothoracic glands. The testis ecdysiotropin (LTE) was isolated from L. dispar pupal brains by a series of high-pressure chromatography steps. Its sequence was Ile-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH, of molecular mass 2,473 Daltons. The predominant signaling pathway for LTE was via G(i) protein, IP3, diacylglycerol and PKC; a modulating pathway, apparently mediated by an angiotensin II-like peptide, was controlled via G(s) protein, cAMP, and PKA. Testis ecdysteroid caused isolated testis sheaths to also synthesize a growth factor that induced development of the male genital tract. The growth factor appeared to be a glycoprotein similar to vertebrate alpha-1-glycoprotein. A polyclonal antibody to LTE indicated LTE-like peptide in L. dispar brain medial neurosecretory cells, the suboesophageal, and other ganglia, and also in its target organ, the testis sheath. LTE immunoreactivity was also seen in testis sheaths of Rhodnius prolixus. LTE-like immunoactivity was also detected in developing optic lobes, antennae, frontal ganglia, and elongating spermatids of developing L. dispar pupae. This may indicate that LTE has a role in development as well as stimulation of testis ecdysteroid synthesis. Published 2001 Wiley-Liss, Inc.

Amino Acid Sequence↗
PubMed2001

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

Amino Acid Sequence↗
PubMed2001

Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport.

The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from approximately 3min in L. maderae to approximately 25min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn-Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph.The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, Neb-TMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes.

Journal Article↗
PubMed2001

Newly discovered functions for some myotropic neuropeptides in locusts.

The field of neuropeptide research in insects during the past twenty years can be characterized by the enormous number of peptides that have been identified. In the locusts, Locusta migratoria and Schistocerca gregaria only, structural information is now available for more than 60 peptides. Quite a number of these peptides were isolated on the basis of their effect on visceral muscle contraction in vitro. A very limited number of reports describe the 'in vivo' function of a myotropic neuropeptide. Moreover, for most of the brain neuropeptides, we ignore whether they have a hormonal function. In this paper, we describe the recently discovered in vivo effects of some of the myotropic peptides, identified in locusts in the past decade. Schistocerca-neuropeptide F accelerates egg development; locustasulfakinin inhibits food intake and [His(7)]-corazonin induces body color pigmentation.

Animals↗
PubMed2001

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata.

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.

Animals↗
PubMed2001

Functional expression of a locust tyramine receptor in murine erythroleukaemia cells.

The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein-coupled receptor. Functional agonist-dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL-TyrLoc). Tyramine elicited a dose-dependent increase of cytosolic Ca2+-ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein-coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.

Animals↗
PubMed2001

cDNA cloning and transcript distribution of two different neuroparsin precursors in the desert locust, Schistocerca gregaria.

Neuroparsins were originally identified in locust corpus cardiacum extracts as folliculostatic or 'antigonadotropic' neuropeptides. This paper presents the cloning of two different neuroparsin precursor cDNAs from the brain of the desert locust, Schistocerca gregaria. The first transcript encodes the precursor (Scg-NPP1) of S. gregaria neuroparsin A and B, whereas the second codes for a novel neuroparsin-related peptide precursor (Scg-NPP2). Both precursors display significant sequence similarities with each other and with the Locusta migratoria neuroparsin (Lom-NPP) and Aedes aegypti ovary ecdysteroidogenic hormone (Aea-OEH1) precursors. Northern blot analysis revealed that these neuroparsin transcripts are present in larval and adult locust brains. Interestingly, the Scg-NPP2 mRNA content proved to be strongly regulated during the reproductive cycle in both adult males and females.

Amino Acid Sequence↗
PubMed2001

Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei.

Identification of substances able to elicit physiological or behavioural processes that are related to reproduction would greatly contribute to the domestication of commercially important crustaceans that do not reproduce easily in captivity. Crustaceans are thought to release urine signals used for chemical communication involved in courtship behaviour. In contrast to insects, very little is known about the endocrinological processes underlying this phenomenon. Therefore, an extract of 3500 central nervous systems of female white shrimp Penaeus vannamei was screened for myotropic activity in order to purify pyrokinin-like peptides that belong to the pyrokinin/PBAN neuropeptide family. Members of this family regulate reproductive processes in insects, including pheromone biosynthesis. Purification of these pyrokinins was achieved by a combination of reversed-phase and normal-phase chromatography. Subsequent characterization by mass spectrometry, Edman degradation and peptide synthesis resulted in the elucidation of two novel peptides. Pev-PK 1 has the primary sequence DFAFSPRL-NH(2) and a second peptide (Pev-PK 2) is characterized as the nonapeptide ADFAFNPRL-NH(2). Pev-PK 1 contains the typical FXPRL-NH(2) (X = G, S, T or V) C-terminal sequence that characterizes members of the versatile pyrokinin/PBAN family. Pev-PK 2 displays an Asn residue at the variable X position of the core pyrokinin sequence. These crustacean pyrokinins are the first to be found in a noninsect. The synthetic peptides display myotropic activity on the Leucophaea maderae as well as on the Astacus leptodactylus hindgut.

Amino Acid Sequence↗