Isoguanosine: isolation from an animal.

Isoguanosine (oxyadenosine or crotonoside), previously known to occur in nature only in the croton bean, was isolated from an animal, the marine nudibranch mollusk Diaulula sandiegensis.

Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin and cholecystokinin.

A sulfated, myotropic neuropeptide termed leucosulfakinin (Glu-Gln-Phe-Glu-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2) was isolated from head extracts of the cockroach Leucophaea maderae. The peptide exhibits sequence homology with the hormonally active portion of the vertebrate hormones human gastrin II and cholecystokinin, suggesting that these peptides are evolutionarily related. Six of the 11 amino acid residues (55 percent) are identical to those in gastrin II. In addition, the intestinal myotropic action of leucosulfakinin is analogous to that of gastrin.

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.

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 beta-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.

Insect satiety: sulfakinin localization and the effect of drosulfakinin on protein and carbohydrate ingestion in the blow fly, Phormia regina (Diptera: Calliphoridae).

Sulfakinins, which are satiety factors in invertebrates, have previously been shown to inhibit feeding in the German cockroach and desert locust. This study examines the occurrence of sulfakinin immunoreactivity and the role of sulfakinin as a feeding satiety factor in the black blow fly, Phormia regina. Specifically, this study examines the effect of sulfakinin on two of the blow fly's nutrient requirements (i.e., carbohydrates and proteins). We observed sulfakinin immunoreactive cells in the brains of both male and female flies. We found that drosulfakinin I (DrmSKI, FDDY[SO(3)H]GHMRFa) significantly inhibited carbohydrate feeding by 44% at the most effective dose (10 nmol) in female flies. Statistically, there was no significant effect on males; however, injections of 10 nmol DrmSKI reduced carbohydrate feeding by 34% compared to the sham. Drosulfakinin had no effect on protein feeding and no significant inhibition was detected in females or males. The results of this study lend further support to the idea that carbohydrate and protein feeding are regulated by separate control mechanisms, especially in Calliphoridae.

Characterization of an RFamide-related peptide orphan GPCR in C. elegans.

We cloned and characterized an orphan FMRFamide-related peptide (FaRP) GPCR in Caenorhabditis elegans. We synthesized numerous structurally different FaRPs that were found in the C. elegans genome by bioinformatic analysis and used them to screen cells expressing the C26F1.6 receptor. Two peptides ending in M(orL)VRFamide elicited a calcium response in receptor-expressing mammalian Chinese hamster ovary cells. The response was dose-dependent and appeared to be very specific; that is, none of the other FaRPs were active, not even closely related peptides also ending in M(orL)VRFamide, which are encoded by the same peptide precursor. Pharmacological profiling with a truncated series of the most active peptide revealed that the full peptide sequence is necessary for receptor activation.

Consensus chemistry and beta-turn conformation of the active core of the insect kinin neuropeptide family.

BACKGROUND: Neuropeptides are examples of small, flexible molecules that bind to receptors and induce signal transduction, thereby eliciting biological activity. The multifunctional insect kinin neuropeptides retain full activity when reduced to only their carboxy-terminal pentapeptide (Phe1-X2-X3-Trp4-Gly5-NH2), thereby allowing extensive structure-function studies and conformational analysis. RESULTS: A combined experimental and theoretical analysis of the insect kinin carboxy-terminal pentapeptide was used to probe the role of each residue, define the bioactive conformation, and design a constrained bioactive analog. Coupling receptor-binding data with two biological activity assays allowed receptor binding and signal transduction to be differentiated. A preferred beta-turn conformation, found for residues 1-4 by molecular dynamics simulations, was tested by designing a conformationally restricted cyclic hexapeptide. This cyclic analog showed a preference for the beta-turn conformation, as shown by a conformational search and nuclear magnetic resonance spectroscopy, and it showed stronger receptor binding but decreased activity relative to highly active linear analogs. CONCLUSIONS: Each residue of the insect kinin carboxy-terminal pentapeptide has a distinct role in conformational preference, specific receptor interactions or signal transduction. The beta-turn preference of residues Phe1-X2-X3-Trp4 implicates this as the bioactive conformation. The amidated carboxyl terminus, required for activity in many neuropeptide families, may be generally important for signal transduction and its inclusion may therefore be essential for agonist design.

Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family.

Two myotropic peptides termed locustatachykinin I (Gly-Pro-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2) and locustatachykinin II (Ala-Pro-Leu-Ser-Gly-Phe-Tyr-Gly-Val-Arg-NH2) were isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence homology is greater with the fish and amphibian tachykinins (up to 45%) than with the mammalian tachykinins. In addition, the intestinal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. The peptides discovered in this study may just be the first in a whole series of substances from arthropod species to be identified as tachykinin family peptides. Moreover, both chemical and biological similarities of vertebrate and insect tachykinins substantiate the evidence for a long evolutionary history of the tachykinin peptide family.

Injection of Dip-allatostatin or Dip-allatostatin pseudopeptides into mated female Diploptera punctata inhibits endogenous rates of JH biosynthesis and basal oocyte growth.

Studies on the catabolism of allatostatins (ASTs) provided the rationale for the design of a series of Dip-allatostatin-derived pseudopeptide mimetic analogues. In vitro, the Dip-ASTs and pseudopeptides show varying degrees of resistance to catabolism and all show significant inhibition of juvenile hormone (JH) biosynthesis. This study was undertaken to determine whether potent Dip-ASTs and/or their pseudopeptide mimetic counterparts caused 'allatostatic' effects in vivo following injection into mated female Diploptera punctata. Animals injected with aqueous solvent or Dip-AST 7(1-7) N-terminal fragment, which excludes the active core region of the ASTs, were used as controls. An in vitro radiochemical assay revealed that injection of Dip-AST 5, 7 or pseudopeptide analogues 397-2 or AST(b)φ2 significantly inhibited the biosynthesis of JH (P<0.05). The results also indicate that basal oocyte growth was significantly inhibited by injection of these same compounds, with the exception of Dip-AST 7 (P<0.05). Analogues 396-1 and 419 did not significantly inhibit rates of JH biosynthesis but did significantly inhibit the growth of basal oocytes. Analyses of feeding, excretion and food absorption/utilization patterns of these same animals suggested that these compounds are not toxic to the insect; rather they directly inhibit the biosynthesis of JH by the corpora allata, and reduce the rate of growth of basal oocytes. Disruption of critical reproductive and/or developmental processes by pseudopeptide analogues of the ASTs could provide novel and selective strategies for future insect pest management.

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Stimulation of alpha-amylase release in the scallop Pecten maximus by the myosuppressins. Structure-activity relationships.

The insect myosuppressin LMS (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2) elicits potent stimulation of the release of the digestive enzyme alpha-amylase from cell suspensions of the stomach-digestive gland complex of the scallop Pecten maximus. The myosuppressins are members of the FMRFamide-like peptide superfamily, which immunocytochemical data confirm is present in the scallop. Structure-activity studies indicated that the two most critical residues for bioactivity are Arg and Phe. Bioactivity of the peptide can be maintained if the basic, aromatic residue His is replaced by another basic residue (Lys) and another aromatic residue (Trp), but not the aromatic Tyr, indicating a sensitivity to the introduction of a phenolic OH group. A restricted-conformation analogue containing a cyclopropyl-Ala residue in position 8 (Cpa-MS) demonstrates an ability to antagonize the amylase secretion activity of LMS at microM concentrations. This result provides evidence that the myosuppressins adopt a tight turn in the C-terminal tetrapeptide active core region while binding to the scallop digestive gland receptor. Cpa-MS may provide a useful tool to neuroendocrinologists studying in vitro and in vivo digestive processes in mollusks and other invertebrates.

Development of amphiphylic mimics of insect neuropeptides for pest control.

Insect neuropeptides regulate virtually all aspects of insect life and are excellent candidates for development of new methods for pest control. However, they do not penetrate insect cuticle and are degraded by enzymes both in the digestive system and hemolymph. We have designed mimics of model neuropeptides by attachment of various lipidic moieties to the amino terminus of the bioactive core of the neuropeptides. These mimics have amphiphylic characteristics that allowed them to penetrate the hydrophobic insect cuticle. The mimics also induced prolonged physiological responses (up to 20 hours) and were resistant to peptidase attack. This knowledge has been used to develop a novel, species-specific approach to insect control.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Synthesis of 3-(12-hydroxy-p-carboranyl)propionic acid, a hydrophobic, N-terminal tyrosine-mimetic for peptides.

A new, p-carborane containing analog of tyrosine, namely 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran(12)-12-yl]propionic acid was prepared from p-carborane in five steps involving hydroxypropylation of O-protected 1-hydroxy-p-carborane as the key transformation. The simple tyrosine mimetic can function as a hydrophobic surrogate for an N-terminal tyrosine residue in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs.

In vitro release of digestive enzymes by FMRF amide related neuropeptides and analogues in the lepidopteran insect Opisina arenosella (Walk.).

The insect neuropeptides FMRF amide, leucomyosupressin (LMS) and neuropeptide analogues leucosulfakinins (FLSK and LSK II Ser (SO(3)H)), perisulfakinin (PSK), proleucosulfakinin (PLSK), 14A[phi1]WP-I, 542phi1, and 378A[5b]WP-I were assayed for their effects on the release of amylase and protease from the midgut tissue of larvae of Opisina arenosella. In the bioassay, empty midgut tubes ligated at both ends using hair were incubated with insect saline containing neuropeptides/analogues in a bioassay apparatus at 37 degrees C for 30 min. After incubation the contents of the midgut preparations were analyzed for amylase and protease activity. In control experiments, the midgut preparations were incubated in insect saline without neuropeptides. The results of the study reveal that for stimulating amylase release from midgut tissue, the peptides require an FXRF amide (X may be methionine or leucine) sequence at the C-terminal. The presence of HMRF amide at C-terminal of peptides may inhibit the release of amylase. Meanwhile, peptides with both FMRF and HMRF amide sequence at the C-terminal are found to be effective in stimulating protease release. The tetrapeptide segment at the C-terminal probably represent the active core of the neuropeptide.

Comparative topical pheromonotropic activity of insect pyrokinin/PBAN amphiphilic analogs incorporating different fatty and/or cholic acid components.

This study presents a comparison of the topical pheromonotropic activity in the tobacco budworm moth of a series of amphiphilic pseudopeptide analogs of the insect pyrokinin/PBAN peptide class incorporating fatty acids of varying chain lengths. While the C16 analog fails to penetrate the moth cuticle, and the C12 only moderately so, shorter chain analogs transmigrate the moth cuticle readily with decreasing cuticle-retention properties. A cholic acid analog topically induces twice the maximal pheromone titer of injected native hormone. From a pest management perspective, these non-aromatic hydrophobic components are expected to be more environmentally benign than benzenoid components previously used in topical insect peptide analogs.

Structural aspects of gastrin/CCK-like insect leucosulfakinins and FMRF-amide.

The leucosulfakinins (LSKs), isolated from head extracts of the cockroach Leucophaea maderae, are sulfated neuropeptides with homology to gastrin and cholecystokinin. The undecapeptide LSK and decapeptide LSK-II stimulate contractions of the isolated cockroach hindgut. Several structural aspects of the two gastrin/CCK-like insect leucosulfakinins (LSKs) and their relation to FMRF-amide are discussed. Replacement of the oxidation sensitive Met residue with isosteric norleucine leads to an analog with retention of biological activity. The Arg residue of the LSKs is critical for cockroach hindgut contractile stimulatory activity, as its introduction into gastrin II transforms the inactive peptide into an active analog. As demonstrated by the equipotent [His14,Arg16]gastrin II, the His8 and Asp5 residues of LSK are not critical for activity. The common C-terminal tetrapeptide of the LSKs ([8-11]LSK) is inactive. Taken together with a comparison of the two LSK structures, the data suggest that the LSK active core resides between [8-11]LSK and [4-11]LSK. This is confirmed by considerable activity displayed by the sulfate analog of LSK-II, which contains an extra sulfate group on the Ser2 residue in the N-terminal region. Homology between the LSKs and molluscan cardioacceleratory and rectum contractile neuropeptide FMRF-amide and Met-enkephalin-Arg6-Phe7 is discussed. The insect LSKs may represent a molecular evolutionary link between the vertebrate gastrin/CCK family and this mammalian enkephalin.

Silkworm diapause induction activity of myotropic pyrokinin (FXPRLamide) insect neuropeptides.

A family of myotropic neuropeptides sharing the common C-terminal pentapeptide Phe-Xxx-Pro-Arg-Leu-NH2 (Xxx = Ser, Thr, Val), known as the pyrokinins, has been isolated from the cockroach Leucophaea maderae and locust Locusta migratoria of the order Orthoptera. A hormone (Bom-DH) that elicits diapause induction in the silkworm Bombyx mori (order Lepidoptera) also contains this C-terminal pentapeptide (Xxx = Gly). The orthopteran pyrokinin neuropeptides elicit significant diapause-inducing activity in the lepidopteran silkworm. Despite containing the sterically bulky, inflexible Val residue in the variable Xxx position, the locust pyrokinin Lom-PK is threefold more active than native Bom-DH as a diapause induction agent. The C-terminally truncated cockroach leucopyrokinin (LPK) fragment, Thr-Ser-Phe-Thr-Pro-Arg-NH2 [LPK(2-7)], proved virtually inactive in the silkworm assay, demonstrating the importance of an intact C-terminal pentapeptide sequence to diapause induction activity. Bom-DH also elicits significant myostimulatory activity in a cockroach hindgut assay, although at a level several orders of magnitude less than the native myotropic peptide LPK. However, the C-terminal pentapeptide of Bom-DH (Xxx = Gly) is equipotent with the LPK C-terminal pentapeptide (Xxx = Thr) as a myostimulatory agent. The cross-activity observed for the various pyrokinins suggests that the receptors that mediate the disparate physiological processes of diapause in the silkworm and hindgut contraction in the cockroach share homologous features.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, characterization and biological activity of a diuretic myokinin neuropeptide from the housefly, Musca domestica.

A competitive ELISA employing a polyclonal antiserum raised against leucokinin-I was used to isolate and purify a myokinin (muscakinin) from 1.05 kg of adult houseflies (Musca domestica). Following solid-phase purification, seven HPLC column steps were used to purify 4.8 nmol of leucokinin-immunoreactive material. Sequence analysis and mass spectrometry were consistent with the structure Asn-Thr-Val-Val-Leu-Gly Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly NH2. This peptide was synthesized and co-eluted with the natural peptide on three different HPLC columns. The activities of natural and synthetic muscakinin were identical, with both producing a 4-5 fold increase in fluid secretion by housefly Malpighian tubules at nanomolar concentrations. The presence of a pair of basic residues (Lys-Lys) suggested muscakinin might be processed further, with the peptide pGlu-Arg-Phe-His-Ser-Trp-Gly NH2 being produced by conversion of an N-terminal glutamine to pyroglutamic acid. However, this analog was 1000-fold less active than the intact peptide, comparable to the activity of AK-V which shares the same C-terminal pentapeptide sequence. The diuretic activity of muscakinin is more than double that of a previously identified CRF-related diuretic peptide (Musca-DP) from the housefly, and the two peptides act synergistically in stimulating fluid secretion. Muscakinin also increased the frequency and amplitude of contractions by housefly hindgut which might further contribute to the excretory process.

Hemolymph and tissue-bound peptidase-resistant analogs of the insect allatostatins.

While neuropeptides of the allatostatin family inhibit in vitro production of juvenile hormone, which modulates aspects of development and reproduction in the cockroach, Diploptera punctata, they are susceptible to inactivation by peptidases in the hemolymph, gut, and bound to internal tissues. Patterns of peptidase cleavage were investigated in two allatostatin analogs in which sterically bulky components were incorporated into the active core region to block peptidase attack. The results were used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both hemolymph and tissue-bound peptidases. This pseudotetrapeptide allatostatin mimetic analog represents a valuable tool to neuroendocrinologists studying mechanisms by which the natural peptides operate and the physiological consequences of challenging an insect with an allatostatin that is not readily degraded via peptidase enzymes. Disruption of critical physiological processes modulated by neuropeptides such as the allatostatins via peptidase-resistant mimetic analogs could form the basis for novel pest insect management strategies in the future.

Comparison of rates of penetration through insect cuticle of amphiphylic analogs of insect pyrokinin neuropeptides.

Rates of penetration through the cuticle of amphiphylic analogs, synthesized by addition of 6-phenylhexanoic acid or 9-fluoreneacetic acid or 1-pyrenebutyric acid to the amino terminus of the pentapeptide Phe-Thr-Pro-Arg-Leu-amide, were assessed by quantitative analysis using reversed phase liquid chromatography. The analogs effectively penetrated the cuticle of both the adult American cockroach and tobacco budworm moth. However, the amounts of analogs that penetrated the cuticle of the cockroach were significantly lower and the rates of penetration were slower than for moth cuticle. Penetration of the analogs through the cuticle was dependent upon the size of the lipidic attachment to the pentapeptide. The 6-phenylhexanoic acid analog penetrated most rapidly followed by the 9-fluoreneacetic acid analog and the 1-pyrenebutyric acid analog penetrated slowest. All of the analogs exhibited an initial rapid period of penetration lasting 2-3 h followed by the establishment of a steady slow release state which lasted between 9-24 h and was dependent upon both the size and surface area of the aromatic lipidic portion of the analog and species of insect to which the analog was applied. The results confirmed the hypothesis that the insect cuticle could be employed as a slow release device for delivery of analogs of insect neuropeptides.