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Hormonal regulation of the shape of identified motoneurons in the moth Manduca sexta.

Motoneurons MN-1 and MN-3 in the moth Manduca sexta go through 3 distinct phases during postembryonic life. During larval life their neuritic fields show allometric growth in concert with larval growth. Larval neurites are then lost during the larval-pupal transition, which is followed by the outgrowth of adult-specific neurites during adult development. In MN-1, the adult-specific arbor typically results from the outgrowth of neurites ipsilateral to the cell body. However, in a small percentage of cells, ipsilateral branches are not present and contralateral branches extend across the midline to fill the vacant space. This altered form of MN-1 is thought to result from the early failure of ipsilateral neurite extension. The steroid hormones, the ecdysteroids, are responsible for the outgrowth of adult neurites. The onset of the latter is correlated with the ecdysteroid increase that promotes adult differentiation and does not occur under conditions such as diapause, in which the normal steroid rise is absent. Artificial replacement of ecdysteroids, however, induces the adult-specific growth. This action of ecdysteroids to cause a change in neuronal form requires the absence of juvenile hormone (JH). Application of JH mimics prior to the onset of the program of adult outgrowth blocks this outgrowth. MN-1 and MN-3 show different times of JH sensitivity, which appear to be correlated with different times of neurite outgrowth. It is concluded that the role of JH is to maintain the status quo of central neurons and prevent changes in form in response to ecdysteroids. In the absence of JH, the ecdysteroids can then exert morphogenetic changes, but the nature of these actions, neurite outgrowth or regression, is likely a function of the developmental history of the cell.

Animals↗

Adipokinetic hormones: cell and molecular biology.

Adipokinetic hormones AKH I (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) and AKH II (pGlu-Leu-Asn-Phe-Ser-Trp-Gly-Thr-NH2) are synthesized by neurosecretory cells (NSC) of the corpora cardiaca (CC) in the locust, Schistocerca gregaria. These NSC constitute a homogeneous 'peptide factory' as each cell synthesizes both AKH I and AKH II. This homogeneity makes the CC an excellent system in which to study aspects of neuropeptide biosynthesis. This report summarizes recent findings on AKH inactivation and metabolism, as well as on AKH prohormone processing and biosynthesis.

Amino Acid Sequence↗

Two puff-specific proteins bind within the 2.5 kb upstream region of the Drosophila melanogaster Sgs-4 gene.

The Drosophila nuclear proteins Bj6 and Bx42 characterized previously are detected in a series of developmentally active puffs on salivary gland chromosomes. Here the binding of both proteins at puff 3C11-12 containing the glue protein gene Sgs-4 is described in more detail. By deletion analysis we show that both proteins bind within a chromosomal segment containing 17-19 kb of DNA surrounding the Sgs-4 gene. They are detectable at this site during the intermoult stages, before the puff regresses in response to the moulting hormone ecdysone. If the Sgs-4 gene together with flanking DNA sequences is brought into a different chromosomal position by P element transfer, both proteins are detected at this new location. Both proteins are bound to the chromosome within the range of 2.5 kb DNA upstream of the Sgs-4 gene. A strain containing a 52 bp deletion within this region fails to bind Bx42 protein suggesting that the missing DNA, which overlaps a hypersensitive region, may be required for the binding of the Bx42 protein.

Animals↗

The ecdysoneless (ecd1ts) mutation disrupts ecdysteroid synthesis autonomously in the ring gland of Drosophila melanogaster.

Ring glands dissected from homozygous l(3)ecd1ts wandering larvae and upshifted in vitro to the restrictive temperature, 29 degrees C, synthesize abnormally low quantities of ecdysteroid. Nevertheless, ecd1 ring glands retain the ability to respond at 29 degrees C to an extract prepared from wild-type larval neural tissues that presumably contain prothoracicotropic hormone (PTTH), although both basal and stimulated levels of synthesis are lower than those in wild-type ring glands. Extracts prepared from ecd1 neural tissue exhibit an unusually high level of PTTH activity. Mutant ring glands downshifted in vitro to the permissive temperature after removal from larvae maintained at 29 degrees C regain the ability to produce normal basal and stimulated ecdysteroid levels. Collectively, these experiments demonstrate that the ecd1 mutation disrupts the physiology of the ring gland at 29 degrees C autonomously and may also interfere with PTTH release.

Animals↗

Hormonal regulation of trehalose metabolism in the blowfly Phormia Regina Meig.: effects of cardiacectomy and allatectomy at the subcellular level.

1. Kinetic properties of adult Phormia fat body glycogen synthetase were studied and compared to other animals. The KM for UDPG is 2.82 mM, decreasing to 0.58 mM in the presence of G-6-P. 2. The specific activity of fat body glycogen synthetase shows a reduction of 30% within 2 days after allatectomy. 3. Fat body T-6-P synthetase activity decreases to 70% of the control value after cardiacectomy. 4. Corpus cardiacum homogenate fails to induce higher T-6-P synthetase activity in cell-free preparations from cardiacectomized flies. 5. Interactions between corpus cardiacum and corpus allatum in regulating carbohydrate metabolism are discussed.

Animals↗

A model for the structure of a homodimeric prohormone: the precursor to the locust neuropeptide AKH I.

We have determined the structure in solution of a homodimeric protein that is a precursor to the locust neuropeptide adipokinetic hormone I using nuclear magnetic resonance spectroscopy. This precursor, called P1, is comprised of two 41 residue strands joined by a single inter-chain disulphide at Cys39. We have also determined the structure of an end product of P1 processing, called APRP1; this is a homodimer comprised of residues 14-41 of P1. Nuclear Overhauser Effect (nOe) data indicate that in both P1 and APRP1, residues 22-37 (numbered with respect to P1) form pairs of alpha-helices, with no evidence for any other secondary structure.

Amino Acid Sequence↗

Hormonal regulation of fat body glycogen phosphorylase activity in larval Manduca sexta during starvation.

The hormonal regulation of fat body glycogen phosphorylase activity in Manduca sexta larvae was studied. During the first 3 hr of starvation the corpora cardiaca (CC) release a glycogen phosphorylase-activating hormone (GPAH). The haemolymph of 24-hr-starved larvae seems to contain increased levels of GPAH, but after 48 hr the titre can be assumed to be as low as prior to starvation. Abdominal stretch receptors do not appear to be involved in the regulation of GPAH release from the CC. Phosphorylase activation can be prevented by the injection of glucose or by feeding the animals with agar containing various carbohydrates. These treatments seem to prevent the release of GPAH from the CC rather than the action of GPAH on the fat body. The physiological signal which initiates peptide release remains unclear.

Animals↗

Eclosion hormone of the silkworm Bombyx mori. Expression in Escherichia coli and location of disulfide bonds.

A gene encoding eclosion hormone (EH) from the silkworm, Bombyx mori was chemically synthesized, inserted into a secretion vector and expressed in Escherichia coli, leading to the production of biologically active EH. Sequence analysis of cystine-containing peptides in a thermolysin digest of this EH established the locations of 3 disulfide bonds in the molecule. Evidence was also obtained that the 6 residues at the NH2-terminal are dispensable but 4 residues at the COOH-terminal play an important role in EH activity.

Amino Acid Sequence↗

FMRFamide- and adipokinetic hormone-like immunoreactivity in the nervous system of the mosquito, Aedes aegypti.

As demonstrated with immunocytochemistry, specific cells and axons in the nervous system of female Aedes aegypti contain antigens immunologically related to FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide) and locust adipokinetic hormone I (AKH). In the supra-esophageal ganglion, including some medial neurosecretory cells, and in all ganglia of the ventral nerve cord, there are 100-120 cells immunoreactive to a FMRFamide antiserum. The same cells cross-react with a bovine pancreatic polypeptide antiserum, but when the latter antiserum is preabsorbed with FMRFamide, immunoreactivity is lost. However, immunoreactivity is maintained when FMRFamide antiserum is preabsorbed with pancreatic polypeptide, suggesting that the immunoreactive peptide is more closely related to FMRFamide. There are 6-12 cells in the supra- and subesophageal ganglia immunoreactive to an AKH antiserum, and some of the same cells are reactive to the FMRFamide antiserum. As well, unpaired cells in each of the abdominal ganglia are positive for both AKH and FMRFamide. Although the function of the FMRFamide- and AKH-like peptides in mosquitoes is unknown, this study, combined with previous reports on the localization of FMRFamide-like peptides in midgut endocrine cells, supports the concept of a brain-midgut neuroendocrine axis in this insect.

Aedes↗

The influence of the brain hormone on retention of blood in the mid-gut of the mosquito Aedes aegypti (L.). III. The involvement of the ovaries and ecdysone.

Most female mosquitoes require a blood-meal in order to produce mature oöcytes. An egg development neurosecretory hormone (EDNH), which is produced in the medial neurosecretory cells (m.n.c.) of the brain and stored in the corpus cardiacum, is released into the haemolymph following the ingestion of blood and is essential for the promotion of ovarian development to maturity. It has been shown that a factor from the m.n.c., presumably EDNH, is necessary if the blood-meal is to be retained in the mid-gut until the oöcytes approach maturity. The present paper shows that retention is not a direct result of the action of EDNH, but is dependent on the ovaries and may well involve ecdysone. Removal of the ovaries before a blood-meal leads to early haem-defaecation, but delay can be restored by injection of ecdysterone. Sub-threshold feeders and mosquitoes decapitated immediately after the intake of blood, each of which would be expected to eliminate the blood-meal early, also show a delay in the onset of haem-defaecation when injected with ecdysterone. Further, both in ovariectomized insects and sub-threshold feeders the time of onset of haem-defaecation is associated with the dose of ecdysterone given.

Aedes↗

Critical period for pupal commitment in the yellow fever mosquito, Aedes aegypti.

Changes in ecdysteroid levels that lead to commitment of pupal and adult development were studied in the yellow fever mosquito, Aedes aegypti. Application of juvenile hormone at the time of pupal commitment usually results in delay or blockage of pupal and adult development. With methoprene, a juvenile hormone mimic, the JH sensitive period was found to be within 19 h after ecdysis to the fourth instar, at which time methoprene treatment caused a delay in pupation. Consistent with this observation, there was a small peak of ecdysteroid levels between 14 and 28 h after ecdysis to the fourth instar. Therefore, the commitment to pupal development occurs most likely between 14 and 19 h after ecdysis to the fourth instar. Levels of transcription of the ecdysone receptor gene show a small peak between 12 and 24 h, and a larger peak between 46 and 66 h after ecdysis to the fourth instar.

Aedes↗

Large-scale purification of prothoracicotropic hormone of the silkworm (Bombyx mori).

Prothoracicotropic hormone has been successfully extracted from as many as 2,800,000 silkworm male adult heads and purified partially through an 8-step procedure. Approximately 7 microgram of the crude preparation ("crude PTTH") shows activity in the debrained Samia pupa test. A further 4-step chromatographic purification of the "crude PTTH" derived from 100,000 Bombyx heads yields 3.4 mg of purified preparation ("highly purified PTTH"), of which 7 ng causes adult development in a debrained Samia pupa.

Animals↗

Characterization of IMP-E3, a gene active during imaginal disc morphogenesis in Drosophila melanogaster.

The steroid hormone 20-hydroxyecdysone (20-HE) induces imaginal discs to form adult appendages in Drosophila. We have isolated a set of six ecdysone-responsive genes that apparently encode disc cell-surface or secreted proteins. Transcripts from one of these genes, IMP-E3, accumulate rapidly within 1-2 h in response to hormone. Developmentally, IMP-E3 transcripts reach maximum levels during the first stages of metamorphosis (white prepupae, WPP) and are primarily limited to imaginal tissues. Transcripts are also present during embryogenesis (0-3 h and 12-18 h). Two different-sized transcripts (1.2 and 1.4 kb) result from differential polyadenylation, with the larger transcript predominating in WPP. The conceptual IMP-E3 protein contains a signal peptide, an RGD sequence, and a potential glycosylphosphatidylinositol anchor. We speculate that the protein provides a transient cue important for imaginal disc morphogenesis.

Amino Acid Sequence↗

Localization of Locusta-DP in locust CNS and hemolymph satisfies initial hormonal criteria.

Locusta-diuretic peptide (Locusta-DP) is a potent stimulant of fluid secretion and cyclic AMP production by locust Malpighian tubules. In this study, a polyclonal antiserum raised to the C-terminus of Locusta-DP reveals a wide distribution of immunoreactive cell bodies and processes throughout the CNS, and endings in two important neurohemal release sites: the corpora cardiaca and the perivisceral organs. HPLC fractionation of CNS, neurohemal structures, and hemolymph reveals immunoreactive material that coelutes with synthetic Locusta-DP and stimulates cyclic AMP production by locust tubules. The identity of the immunoreactive and biologically active material is confirmed as authentic Locusta-DP by mass spectrometry.

Animals↗

The possible evolutionary significance of repeat elements near and within an early chorion gene in the late chorion locus of Bombyx mori.

Three beta-type early chorion gene copies (6F6.1, 6F6.2, and 6F6.3) are dispersed in the late region of chorion locus Ch1-2. Detailed analysis of the 5'-flanking region and the intron of 6F6.1 shows that they contain sequences that are homologous to Bombyx mori Bm1 repeat elements. Interestingly, the Bm1-type segment of the intron is interrupted by the insertion of a sequence that shows significant similarities with part of an intron of B. mori and Bombyx mandarina fibroin genes, and with part of the 3'-flanking region of B. mori prothoracicotropic hormone and tRNA-Glu genes; this sequence may represent a new repetitive, possibly transposable, element of B. mori. Following the Bm1-homologous sequence of the 6F6.1 5'-flanking region and preceding the gene promoter region, a short DNA segment shows sequence motifs that are also present in the ErA.1 promoter region. The occurrence of these sequences near one end or within the Bm1 repeat element is suggestive of complex sequence transfer events. Comparative analysis of known B. mori chorion alpha-gene promoters and of Bm1 repeat elements suggests, with marginal statistical significance, that these two sets of sequences contain common elements.

Animals↗

Preparation of a specifically tritiated locust adipokinetic hormone analog with full biological potency.

A synthetic peptide related to locus adipokinetic hormone ( AKH ) and shrimp red pigment concentrating hormone ( RPCH ) containing a tyrosine residue in place of phenylalanine was iodinated and the 3,5- diiodotyrosyl derivative was isolated by reverse phase HPLC. Catalytic dehalogenation of the diiodo derivative in the presence of tritium yielded the tritiated AKH analog which was isolated by gel filtration on Sephadex LH-20 and reverse phase HPLC. The tritiated peptide was formed to be identical to AKH in its ability to stimulate lipid release into the hemolymph of locusts in vivo where the diiodotryrosyl derivative was inactive. The specific radioactivity of the tritiated peptide was 57.2 Ci/mmol, or 99% of the theoretical value.

Animals↗

cDNA cloning of a mandibular organ inhibiting hormone from the spider crab Libinia emarginata.

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crab Libinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks of Libinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.

Amino Acid Sequence↗

Developmental profile of the changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx mori: correlation with ecdysteroid secretion.

A very sensitive time-resolved fluoroimmunoassay for the prothoracicotropic hormone (PTTH) of the silkworm Bombyx mori has been established. The lower limit of detection in this assay was 0.1 pg. With this assay method, the amounts of PTTH in the central nervous system and hemolymph were quantified. PTTH was detected only in the brain within the central nervous system, and, in the fifth instar, its content in the brain increased gradually with larval growth and decreased rapidly after the beginning of wandering. A substantial amount of PTTH was also found in the retrocerebral complex of day-3 fifth instar larvae, accounting for 28% of total PTTH. The PTTH titer in hemolymph changed dramatically during Bombyx development, with a small peak in the middle of the fourth instar, medium-sized peaks at the wandering and prepupal stages in the fifth instar, and a large prolonged peak during early pupal-adult development. The changes were overall closely correlated with those in hemolymph ecdysteroid titer. However, some unexpected aspects of PTTH dynamics in hemolymph have also been disclosed. Based on these observations, the significance of PTTH secretion in the control of insect development is discussed.

Animals↗