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Growth Promotion in Pea Stem Sections. III. By Alkyl Nitriles, Alkyl Acetylenes and Insect Juvenile Hormones.

C(14), C(15), and C(16) alkyl nitriles, and C(16) and C(18) alkyl acetylenes at 10 to 105 micromolar concentrations promote the growth of stem sections from red-light-exposed seedlings of dwarf peas (Pisum sativum L. cv. Progress No. 9). Similar results were obtained with substances active as insect juvenile hormones, namely farnesol, the racemic ethyl ester of 1 of the natural hormones, and a "synthetic juvenile hormone" mixture, the latter 2 having as high an activity in the pea assay as any lipid reported previously. A sterically nearly identical compound, methyl-RS-10,11-epoxyfarnesoate, is a weak insect hormone and did not promote plant growth. Thus activity in peas and in insects is in some cases parallel. Other similarities and some differences are discussed. Peas appear to require molecules longer than 20A, while insect activity is maximal at that length. All active molecules are ineffective in promoting pea stem elongation by themselves, indole acetic acid must also be present. The lipid effect in plants and the juvenile hormone response in insects have much in common and the evidence suggests they could have a similar locus of action in a membrane controlling respiratory function.

Journal Article↗

A new approach in integrated control: insect juvenile hormone plus a hymenopteran parasite against the stable fly.

Two insect juvenile hormone analogs, 4[(6,7-epoxy-3-ethyl-7-methyl-2-nonenyl)oxy] benzene and 6,7-epoxy-1-(p-ethylphenoxy)-3,7-dimethyl-2-octene, when applied topically to pupae of the stable fly, Stomoxys calcitrans (L.), were morphogenetically effective against the metamorphosing pupae but did not affect oviposition and development of the hymenopteran parasite, Muscidifurax raptor Girault and Sanders, in the treated pupae. Also, reproductivity of the parent generation of parasites was not affected.

Animals↗

Radioimmunoassay of insect juvenile hormones and of their diol derivatives.

We have developed a radioimmunoassay for insect juvenile hormones. The C18 hormone (JH I) was first converted into diol by opening the 10, 11-epoxy ring. Diol was then succinylated and coupled to human serum albumin to make it immunogenic. High titer antisera were obtained from immunized rabbits. Succinyl juvenile hormone was also coupled to the peptide glycyltyrosine and iodinated so as to form a water-soluble 125I-labelled analogue well recognized by antibodies (60% bound by the 1/2000 000 dilution of antiserum routinely used in radioimmunoassay). Standards and biological samples were treated with acidic dioxane in order to convert each hormone into its corresponding diol. In this way, the sensitivity threshold of the radioimmunoassay was under 0.015 pmol. All three diols were equally recognized by the antibodies. Hormones JH I, JH II and JH III could be assayed separately as diols after thin-layer chromatography or high-pressure liquid chromatography purification of the biological samples. This method was used to determine physiological levels of juvenile hormones in the haemolymph of several insects at different development stages including embryos and in corpora allata cultures.

Animals↗

Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate.

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and 1-oleyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate and diolein are inactive. The inhibitory effect of phorbol 12-myristate 13-acetate is fully reversed by 2E,6E-farnesol or by 2E,6E-farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20-hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.

Animals↗

Insect prothoracicotropic hormone: evidence for two molecular forms.

In an insect, the tobacco hornworm Manduca sexta, the cerebral neuropeptide prothoracicotropic hormone (PTTH), the primary effector of postembryonic development, exists as two molecular forms. These two PTTH's elicit characteristic in vitro dose responses of activation of prothoracic glands from different developmental stages, an indication that during development the glands change in their sensitivity to the neurohormones. Both PTTH's are active in a specific in situ bioassay. Since they may be released in situ at stage-specific times to evoke distinctly different developmental responses, the PTTH neuroendocrine axis appears to be an effective system for determining the functions of molecular forms of a neurohormone in the regulation of growth and development.

Animals↗

Insect adipokinetic hormones: release and integration of flight energy metabolism.

Insect flight involves mobilization, transport and utilization of endogenous energy reserves at extremely high rates. Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine cells, integrate flight energy metabolism. The complex multifactorial control mechanism for AKH release in the locust includes both stimulatory and inhibitory factors. The AKHs are synthesized continuously, resulting in an accumulation of AKH-containing secretory granules. Additionally, secretory material is stored in large intracisternal granules. Although only a limited part of these large reserves appears to be readily releasable, this strategy allows the adipokinetic cells to comply with large variations in secretory demands; changes in secretory activity do not affect the rate of hormone biosynthesis. AKH-induced lipid release from fat body target cells has revealed a novel concept for lipid transport during exercise. Similar to sustained locomotion of mammals, insect flight activity is powered by oxidation of free fatty acids derived from endogenous reserves of triacylglycerol. However, the transport form of the lipid in the circulatory system is diacylglycerol (DAG) that is delivered to the flight muscles associated with lipoproteins. While DAG is loaded onto the multifunctional insect lipoprotein, high-density lipophorin (HDLp) and multiple copies of the exchangeable apolipoprotein III (apoLp-III) associate reversibly with the expanding particle. The resulting low-density lipophorin (LDLp) specifically shuttles DAG to the working muscles. Following DAG hydrolysis by a lipophorin lipase, apoLp-III dissociates from the particle, regenerating HDLp that is re-utilized for lipid uptake at the fat body cells, thus functioning as an efficient lipid shuttle mechanism. Many structural elements of the lipoprotein system of insects appear to be similar to their counterparts in mammals; however, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different.

Animals↗

Insect prothoracicotropic hormone: a new member of the vertebrate growth factor superfamily.

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear-cut sequence similarity to any other proteins has not been observed. By disulfide-bond pattern analysis and modeling of the PTTH structure based on the known three-dimensional (3D) structures of growth factor family with cystine-knot motif, we propose that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, beta-nerve growth factor (beta-NGF), transforming growth factor-beta 2 (TGF-beta 2), and platelet-derived growth factor-BB (PDGF-BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, beta-NGF, TGF-beta 2 and PDGF-BB.

Amino Acid Sequence↗

A partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity.

A partition assay was developed to measure insect juvenile hormone (JH) I and III metabolism in biological samples containing both JH esterase and JH epoxide hydrolase activity. The assay utilizes commercially available radiochain 3H-labeled JH as substrate and the selective JH esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone. JH partitions into an isooctane phase and the metabolites JH acid, JH diol, and JH diol-acid into aqueous methanol after incubation of JH substrate with inhibited and uninhibited sample. The assay provides a time- and cost-efficient alternative to the currently available thin-layer chromatography method for the measurement of JH esterase and epoxide hydrolase activity.

Acetone↗

The activity of insect juvenile hormone mimics in larval Amblyomma hebraeum Koch (Acarina: Metastriata: Ixodidae).

A total of 14 insect juvenile hormone mimics was tested for activity in Amblyomma hebraeum by exposing newly engorged larvae to filter paper impregnated with hormone mimics. The most active compounds used in this assay were HS 103 (6-ethyl-3-pyridyl geranyl ether; EC50=0,0018 mg/cm2), ZR 512 (Ethyl 3,7,11-trimethyldodeca-2,4-dienoate; EC50=0,0022 mg/cm2), HS 2 (6-methyl-3-pyridyl geranyl ether; EC50=0,0035 mg/cm2), ZR 615 (N-ethyl 3,7,11-trimethyldodeca-2,4-dieneamide; EC50=0,0035 mg/cm2), ZR 777 (Prop-2-ynyl 3,7,11-trimethyldodeca-2,4-dienoate; EC50=0,0039 mg/cm2) and ZR 515 (Isopropyl-11-methoxy 3,7,11-trimethyldodeca-2,4-dienoate; EC50=0,0094 mg/cm2). Activity in this assay was similar to that reported in insects and was consistent with the susceptibility of these compounds to metabolic inactivation. The results suggest that ZR 615 may be of possible use in tick control.

Animals↗

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry.

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor-urotensin-sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of approximately 1 microM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29-R30 and R30-A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

Amino Acid Sequence↗

Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria.

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.

Aldose-Ketose Isomerases↗

Biosynthesis and degradation of the arginine-vasopressin-like insect diuretic hormone, a neurohormone in the migratory locust.

The migratory locust (Locusta migratoria) contains two neuropeptides structurally related to mammalian arginine-vasopressin: a 9-residue monomer, without known biological activity, and its antiparallel dimer: the arginine-vasopressin-like insect diuretic hormone which increases urine production at the Malpighian tubules level. We demonstrate hereunder that a transformation monomer-to-dimer-to a degradative product occurs in several steps. (1) A transformation monomer-to-dimer takes place in the suboesophageal ganglion, the site of biosynthesis of the monomer, obviously enzymatically controlled. (2) Monomer and dimer are simultaneously released from the suboesophageal ganglion into the haemolymph where the transformation monomer-to-dimer continues. (3) Dimer is then transported to its target-tissue, the Malpighian tubules, where it is degradated by another enzymatic process.

Animals↗

Analysis and quantitation of insect juvenile hormones using chemical ionization ion-trap mass spectrometry.

A method for identification and quantitation of insect juvenile hormones (JH) has been developed using capillary gas chromatography-chemical ionization (isobutane)-ion-trap mass spectroscopy. The method does not require derivatization of samples or use of selected ion monitoring. Analysis over a mass range of 60-350 u allowed for identification of as little as 0.01 pmol of individual JH homologs. Quantitative analysis was based on the ion intensities of six diagnostic ions and the summed intensities of these ions for each homolog. The ratio of diagnostic ions did not vary significantly over a range of concentrations from 2.7 to 200 pg. The technique was used to identify and quantify the amounts of JH homologs secreted by individual retrocerebral complexes from the moth Manduca sexta maintained in tissue culture and to identify JH III from hexane extracts of hemolymph of the Caribbean fruit fly. No discrimination due to disparate abundance ratios of the individual homologs was found when analyzing natural product samples differing in concentration by at least fivefold. The technique allows for facile, concrete identification and quantitation of biologically relevant amounts of JH. The ability to analyze samples without derivatization or fractionation by chromatographic methods, coupled with data acquisition over a broad mass range, provides levels of accuracy and confidence greater than those of other methods.

Animals↗

Circadian orchestration of developmental hormones in the insect, Rhodnius prolixus.

This review presents a new perspective on the circadian regulation and functions of insect developmental hormones. In Rhodnius prolixus (Hemiptera), the brain neuropeptide prothoracicotropic hormone (PTTH) is released with a circadian rhythm that is controlled by paired photosensitive clocks in the brain. These clocks comprise the dorsal and lateral PER/TIM clock neurons known to regulate behavioral rhythms in Drosophila. Axons of PTTH and clock cells make close contact. Photosensitive PER/TIM clocks also reside in the paired prothoracic glands (PGs), which generate rhythmic synthesis and release of the ecdysteroid molting hormones. The PG clocks are entrained by both light and PTTH. These four clocks are coupled together by both nerves and hormones into a timing system whose primary regulated output is the circadian rhythm of ecdysteroids in the hemolymph. This complex timing system appears necessary to ensure circadian organization of the gene expression that is induced in target cells by ecdysteroids via circadian cycling of the nuclear ecdysteroid receptor (EcR). This multioscillator system serves to transduce 'the day outside' into endocrine rhythms that orchestrate 'the day inside'. It has many functional similarities with vertebrate circadian systems.

Animals↗

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.

Animals↗