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PubMed2005

Regulation of capacitative Ca2+ entry by prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori.

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.

Animals↗
PubMedDate not supplied

Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L. ): developmental changes in sensitivity to JH precursor and allatotropin.

Juvenile hormone (JH) is considered the prime endogenous signal for the induction of queen development in honey bees (Apis mellifera L.). At the beginning of the last (5th) larval stadium, worker corpora allata synthesize less JH than queen corpora allata as a consequence of a limited production of JH precursors and a caste- and stage-specific block of the terminal step in JH biosynthesis. As previously shown, the Manduca sexta allatotropin stimulates JH biosynthesis in honey bee corpora allata in a dose-dependent and reversible manner, but can not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for worker early 5th instars. In experiments with M. sexta allatotropin and with the JH precursor farnesoic acid, we found characteristic stage-specific differences in their effects on JH biosynthesis. From the end of the spinning stage on, corpora allata could be stimulated by farnesoic acid to a much higher extent than in earlier developmental stages, suggesting a sudden increase in epoxidase activity. Manduca sexta allatotropin, however, stimulated corpora allata activity until the end of the spinning stage, at which time the corpora allata become suddenly insensitive. These data suggest that in worker larvae, important changes in the regulation of the terminal enzymatic steps in JH biosynthesis occur at the transition from the spinning stage to the prepupal stage. However, the analysis of in vitro activities of the involved enzymes, O-methyltransferase and methyl farnesoate epoxidase, remained inconclusive.

Animals↗
PubMed1986

The physiology of wandering behaviour in Manduca sexta. IV. Hormonal induction of wandering behaviour from the isolated nervous system.

Prior to exposure to ecdysteroids, the isolated central nervous system (CNS) of the fifth instar Manduca sexta larvae exhibited infrequent motor bursts over a 24-h period of extracellular recording from segmental motor nerves. In contrast, the CNS isolated from wandering larvae was characterized by persistent, frequent spontaneous motor bursts throughout the 24-h incubation. The motor bursts generated by the isolated CNS of wandering larvae were similar to those observed in deafferented segments of partially dissected wandering larvae during locomotion. In both cases bursts in the deafferented ganglia were synchronous and had a lower frequency than in intact animals. Removal of the brain from a CNS isolated prior to ecdysteroid exposure resulted in the appearance of spontaneous bursts, which were abolished by removing the suboesophageal ganglion (SEG). By contrast, when the brain was removed from the isolated CNS of wandering larvae, spontaneous bursts ceased. These results parallel the behavioural effects of the same lesions in intact larvae of the respective stages. The CNS isolated from larvae prior to ecdysteroid exposure initiated sustained frequent bursting after an average latency of 15 h following incubation in haemolymph taken from larvae during the interval of ecdysteroid secretion. Incubations of the CNS with 1 microgram ml-1 20-hydroxyecdysone (20-HE) resulted in the onset of the same pattern of sustained motor activity. In a CNS isolated prior to ecdysone release, it was necessary and sufficient to expose the brain to 20-HE in order to induce the state of persistent motor bursts characteristic of wandering. We conclude that the spontaneous persistent motor bursts observed in the isolated CNS of wandering larvae are directly related to the sustained locomotion seen during the wandering behaviour. 20-HE acts directly on the CNS, specifically the brain, to induce this state of neural activity.

Animals↗
PubMed2005

Molecular characterization and expression of prothoracicotropic hormone during development and pupal diapause in the cotton bollworm, Helicoverpa armigera.

Using a strategy of rapid amplification of cDNA ends, the cDNA encoding prothoracicotropic hormone (PTTH) was cloned from the brain of Helicoverpa armigera (Hearm). The Hearm-PTTH cDNA contains an open reading frame encoding a 226-amino acid preprohormone, which shows high identity with the closely related noctuid moths, Helicoverpa zea (98%) and Heliothis virescens (94%), and low identity with five species of Bombycoidea: Bombyx mori (57%), Manduca sexta (55%), Hyalophora cecropia (52%), Samia cynthia ricini (49%) and Antheraea peryni (48%). Hearm-PTTH cDNA shares important structural characterization known from other PTTHs, such as seven cysteine residues, proteolytic cleavage site, glycosylation site, and hydrophobic regions within the mature peptides. Northern blot analysis indicated a 0.9kb transcript present only in the brain. Using the more sensitive technique of RT-PCR, PTTH mRNA was also detected in the subesophageal ganglion, thoracic ganglion, abdominal ganglion, midgut and fat body. During the pupal stage, PTTH mRNA in the brain remained at a constant high level in nondiapausing individuals, was low in diapausing pupae, but increased again at diapause termination. The PTTH protein was detected only in the brain by Western blot analysis. Immunocytochemical results revealed that Hearm-PTTH is localized in two pairs of dorsolateral neurosecretory cells within the brain. Recombinant Hearm-PTTH was successfully expressed in E. coli, and purified recombinant-PTTH was effective in breaking pupal diapause. The results are consistent with a role for PTTH in the regulation of diapause in this species.

Amino Acid Sequence↗
PubMed1994

Cloning, characterization, and genetics of the juvenile hormone esterase gene from Heliothis virescens.

The gene for juvenile hormone esterase (JHE) was cloned from Heliothis virescens (Lepidoptera: Noctuidae). A genomic library was constructed from embryonic DNA and screened with a homologous N-terminal probe from the JHE cDNA. Five genomic clones were isolated and analyzed by dot blot hybridization using regions of the JHE cDNA as probes. Clone C hybridized to both 5' and 3' probes from the JHE cDNA, suggesting that clone C contains both ends of JHE gene. This was verified by sequencing the ends of the JHE gene from clone C using primers from both the 5' and 3' ends of the JHE cDNA. Additional sequencing and restriction mapping were used to characterize the gene. The gene is c. 8 kb long and contains four introns with consensus intron-exon junctions. One of the introns is relatively large (4 kb) and is situated near the extreme 5' end of the gene. Genetic analysis of RFLP variation in interspecific and intraspecific crosses shows that the JHE locus is single-copy with no closely related paralogs and is autosomally encoded in Heliothis. Therefore the developmental pattern of expression of this gene and the previously documented sequence variation in cDNA clones is not explainable by reference to a JHE gene family with distinct structural loci for the different forms.

Animals↗
PubMed1994

Polymorphism and linkage analysis of the prothoracicotropic hormone gene in the silkmoth, Bombyx mori.

We looked for polymorphism of the prothoracicotropic hormone gene locus (Ptth) among inbred strains of the silkmoth, Bombyx mori, by in vitro DNA amplification (polymerase chain reaction), and found three alleles, PtthA, PtthB and PtthC. The PtthA allele contained a third intron consisting of 680 bp and a fourth intron of 350 bp. PtthB contained the same size third intron but a longer fourth intron of 490 bp, while PtthC had a longer third intron of 1080 bp and a shorter fourth intron of 350 bp. In 29 strains which we examined, 9 strains had PtthA, 8 strains had PtthB, and 2 strains had PtthC. The other 10 strains had heterogeneous genotypes with the same 3 alleles. Linkage analyses using these polymorphisms showed that Ptth was linked to the or gene on chromosome 22, and was independent of other chromosome markers. To determine the map position, we obtained an F1 hybrid between the w41 strain (PtthC+or+mw) and the w22 strain (PtthB or mw), and backcrossed the F1 hybrid to females of the w22 strain. From the segregation of or, mw and Ptth in 861 progeny, recombination values were calculated as 18.5% between or and mw, 23.9% between mw and Ptth, and 7.3% between Ptth and or. Because or and mw have already been mapped at positions 22-8.9 cM and 22-25.2 cM, respectively, we mapped Ptth at 22-2.5 cM. The Ptth locus is independent of any loci which are known to control growth and molting.

Animals↗
PubMed1984

In vitro activity of dipteran ring glands and activation by the prothoracicotropic hormone.

The relationship between hemolymph ecdysteroid titer, ring gland (RG) activity, and prothoracicotropic hormone (PTTH) activation of RG in vitro has been examined during the postfeeding larval, prepupal, and pupal stages of Sarcophaga bullata. Using the ecdysteroid radioimmunoassay (RIA), two significant peaks were recorded during the red spiracular stage and during the first few hours after the formation of the white prepupa and a third large peak 9 hr later. It is postulated that these increases in ecdysteroid titer are involved in the processes of pupariation, puparial tanning, and pupation, respectively. Ring glands isolated from Sarcophaga of known ages were incubated in vitro and the secreted ecdysone was quantified by RIA. Ring glands from early red spiracular stage larvae proved to be the most active and subsequent secretory activity of the RG oscillated every 4 hr with the oscillations gradually decreasing in amplitude. RG activity returned to a basal level 24 hr after formation of the white prepupa, about the time that the hemolymph ecdysteroid titer fell to its basal level. To demonstrate PTTH activity in vitro, brains from 3- to 4-hr prepupae were chosen to activate ring glands from postfeeding larvae. Using a graded series of dilutions of PTTH extract it was shown that a dose-response relationship could be obtained for Sarcophaga similar to that demonstrated or the Manduca sexta PTTH-prothoracic gland system. In Sarcophaga maximal activation resulted in a 10-fold increase in ecdysone synthesis and secretion by ring glands stimulated with 0.5 brain eq. Half-maximal stimulation was attained with 0.2 brain eq of PTTH extract.

Animals↗
PubMed1994

Changes in general and specific protein synthesis that accompany ecdysteroid synthesis in stimulated prothoracic glands of Manduca sexta.

The prothoracic glands of fifth instar Manduca sexta larvae respond to stimulation by the brain neuropeptide, prothoracicotropic hormone (PTTH), with a several-fold increase in the rate of ecdysteroid synthesis. Previous studies have shown that this response requires protein synthesis and that the action of PTTH can be mimicked by dibutyryl cAMP (dbcAMP) and the Ca2+ ionophore, A23187. To further understand the role of protein synthesis in the response of prothoracic glands to PTTH, patterns of protein synthesis in stimulated glands were examined using glands incubated in vitro with [35S]methionine. All three agents caused an increase in the rate of ecdysteroid synthesis as well as an increase of up to 300% in the synthesis and/or accumulation of three proteins (p100, p70, and p"50") within 2 h of stimulation. Changes in these three proteins were specific to the prothoracic gland, were not elicited by non-brain peptides and were not simply a result of increased general protein synthesis in the gland. Exposure of the glands to A23187 alone, or concurrently with dbcAMP, resulted in increased synthesis of p100, p70, p"50" and ecdysteroids but decreased general protein synthesis. Increased synthesis of these proteins could be detected within 15 min after initiating PTTH stimulation. The behavior of these three proteins makes them candidates for modulators of ecdysteroid synthesis in the prothoracic gland. The results suggest also that PTTH may activate two biochemical pathways in the gland: one path leading to increased synthesis of the p100, p70, and p"50" proteins and increased ecdysteroid synthesis, and the second leading to increased general protein synthesis. This second trophic effect is vulnerable to intracellular Ca2+ changes that do not inhibit the first pathway.

Animals↗
PubMed1990

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta.

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor--allatinhibin (AI)--from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 microliters medium. CA must be exposed to AI for 12-16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.

Animals↗
PubMed2003

Stimulation of JH biosynthesis by the corpora allata of adult female Aedes aegypti in vitro: effect of farnesoic acid and Aedes allatotropin.

Previous studies have demonstrated that the synthesis of juvenile hormone (JH) by the isolated corpora allata (CA) complex in vitro as well as the JH titer in the yellow fever mosquito Aedes aegypti are elevated before feeding and low after a blood meal. In the present study, we used an in vitro radiochemical assay to analyze the effect of farnesoic acid (FA) and Aedes allatotropin (Aedes-AT) on the biosynthesis of JH and methyl farnesoate (MF) by the isolated CA complex of A. aegypti adult female. CA complex from day-0 females (0-1 h after emergence) exhibited a low basal juvenile hormone III (JH III) biosynthetic activity and did not respond to either allatotropic or FA stimulation. However, incubation of CA complexes from newly emerged females with Aedes-AT plus FA resulted in very high production of JH III. This is the first report suggesting that allatotropin makes corpora allata in newly emerged females capable for JH biosynthesis. When we studied CA complexes dissected from females 1 day after emergence, the stimulatory action of Aedes-AT was strong and dose-dependent, with maximum stimulation in the range of 10(-8)-10(-9) mol l(-1), suggesting that Aedes-AT is indeed a true allatotropin (a molecule with allatotropic activity) in A. aegypti. The addition to the culture medium of 40 micro mol l(-1) FA, a JH precursor, resulted in a 9-fold increase in JH III biosynthesis in 2-, 4- and 6-day-old sugar-fed females. The two major labeled products synthesized by the stimulated CA complex were identified as JH III and MF by RP-HPLC and GC-MS. Treatment of CA complexes with FA, but not Aedes-AT, resulted in an increase in MF. Application of both Aedes-AT and FA to the CA complexes of 2-, 4- and 6-day-old females resulted in the same effects as FA alone. These data suggest that in sugar-fed females, FA and Aedes-AT exert different effects on the terminal steps in JH biosynthesis.

Aedes↗
PubMed2005

Establishment of a sandwich ELISA system to detect diapause hormone, and developmental profile of hormone levels in egg and subesophageal ganglion of the silkworm, Bombyx mori.

In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

Animals↗
PubMed2005

Mosquito natriuretic peptide identified as a calcitonin-like diuretic hormone in Anopheles gambiae (Giles).

Mosquito natriuretic peptide (MNP), an uncharacterised peptide from the yellow fever mosquito, Aedes aegypti, acts via cyclic AMP to stimulate secretion of Na+-rich urine by opening a Na+ conductance in the basolateral membrane of Malpighian tubule principal cells. Corticotropin releasing factor (CRF)-related peptides and calcitonin (CT)-like diuretic peptides use cyclic AMP as a second messenger and were therefore considered likely candidates for MNP. BLAST searches of the genome of the malaria mosquito Anopheles gambiae, gave sequences for the CRF-related peptide Anoga-DH44 and the CT-like peptide Anoga-DH31, which were synthesised and tested for effects on Malpighian tubules from An. gambiae and Ae. aegypti, together with 8-bromo-cyclic AMP. The cyclic AMP analogue stimulated secretion of Na+-rich urine by An. gambiae Malpighian tubules, reproducing the response to MNP in Ae. aegypti. It also depolarised the principal cell basolateral membrane voltage (Vb) while hyperpolarising the transepithelial voltage (Vt) to a similar extent. Anoga-DH4) and Anoga-DH31 stimulated production of cyclic AMP, but not cyclic GMP, by Malpighian tubules of An. gambiae. Both peptides had diuretic activity, but only Anoga-DH31 had natriuretic activity and stimulated fluid secretion to the same extent as 8-bromo-cyclic AMP. Likewise, Anoga-DH31 reproduced the effects of cyclic AMP on tubule electrophysiology, whereas Anoga-DH44 initially hyperpolarised Vb and depolarised Vt, which is the opposite of the effect of Anoga-DH31. Anoga-DH44 and Anoga-DH31 were also tested for effects on fluid secretion and ion transport by Ae. aegypti tubules. As in An. gambiae, the CRF-related peptide Anoga-DH44 had a non-specific effect on the transport of Na+ and K+, whereas the CT-like peptide Anoga-DH31 specifically stimulated transepithelial Na+ transport. We conclude that the CT-like peptide Anoga-DH31 is the previously uncharacterised mosquito natriuretic peptide.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti.

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.

Aedes↗
PubMed1998

The knirps and knirps-related genes organize development of the second wing vein in Drosophila.

The neighboring homologous knirps (kni) and knirps-related (knrl) genes in Drosophila encode transcription factors in the steroid hormone receptor superfamily. During early embryogenesis, kni functions as a gap gene to control expression of segmentation genes within the abdominal region of the embryo. In this study, we present evidence that kni and knrl link A/P positional information in larval wing imaginal discs to morphogenesis of the second longitudinal wing vein (L2). We show that kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. The kni and knrl L2 stripes abut the anterior border of the broad central expression domain of the Dpp target gene spalt major (salm). We provide evidence that radius incompletus (ri), a well-known viable mutant lacking the L2 vein, is a regulatory mutant of the kni/knrl locus. In ri mutant wing discs, kni and knrl fail to be expressed in the L2 primordium. In addition, the positions of molecular breakpoints in the kni/knrl locus indicate that the ri function is provided by cis-acting sequences upstream of the kni transcription unit. Epistasis tests reveal that the kni/knrl locus functions downstream of spalt major (salm) and upstream of genes required to initiate vein-versus-intervein differentiation. Mis-expression experiments suggest that kni and knrl expressing cells inhibit neighboring cells from becoming vein cells. Finally, kni and knrl are likely to refine the L2 position by positively auto-regulating their own expression and by providing negative feedback to repress salm expression. We propose a model in which the combined activities of kni and knrl organize development of the L2 vein in the appropriate position.

Animals↗
PubMed1989

Ionic dependence of depolarization-mediated adipokinetic hormone release from the locust corpus cardiacum.

The locust corpus cardiacum (CC) is a peripheral neurohemal organ in which are clustered a prodigious array of neurosecretory cells (NSCs), nearly all of which synthesize and release adipokinetic hormones (AKHs). We have examined the extracellular requirements for Na+ and Ca2+ in the process of AKH release following NSC depolarization by high extracellular K+ or veratridine. Na+ is not required for release mediated by high external K+ although Ca2+ is. The Ca2+ channel antagonists cobalt and lanthanum prevent release and support the hypothesis that depolarization with K+ leads to Ca2+ channel activation and subsequent AKH release. Tetrodotoxin does not block K+-mediated release suggesting that Na+ channel activation and Na+ influx are not required for K+-mediated release. The alkaloid veratridine leads to cobalt- and tetrodotoxin-sensitive release and this suggests that cell depolarization by Na+ channel activation is nevertheless capable of opening Ca2+ channels and initiating release. Release mediated by high external K+ is reduced by nifedipine but is not significantly reduced by methoxyverapamil, however veratridine-mediated release is slightly reduced by methoxyverapamil. Glandular lobes accumulated greater amounts of 45Ca2+ following high K+-mediated depolarization compared to glands incubated in normal saline and this enhanced accumulation was blocked by the Ca2+ channel antagonist lanthanum. During prolonged exposure to high K+ saline the release of AKHs and the uptake of 45Ca2+ reach a maximum and then gradually decline. The temporal pattern of the reduction in AKH release is similar to that of 45Ca2+ accumulation by the glandular lobe. This reduction in AKH release and 45Ca2+ uptake may result from inactivation of Ca2+ channels associated with the release process. These results indicate that Ca2+ influx into the NSCs by way of voltage-sensitive Ca2+ channels plays a critical role in the process of depolarization-mediated AKH release.

Animals↗
PubMed1991

Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands.

When a pair of prothoracic glands (PGs) were removed from Manduca sexta pupae on the day of pupation, the hemolymph ecdysteroid titer remained at a low level. When a portion of the gland pair was extirpated from pupae after the critical period for prothoracicotropic hormone release, the maximum hemolymph ecdysteroid titer was reduced in proportion to the mass of the PGs removed. These findings clearly showed that the PGs in intact pupae are responsible for the elevated ecdysteroid titer required to elicit adult development on schedule. When brains were removed on the day of pupation, the initiation of adult development was delayed for weeks or months. In contrast, pupae whose PGs were removed on the day of pupation initiated development only 7 days late, indicating the existence of an additional source of pupal ecdysteroids. Further, abdomens of male M. sexta that were isolated on the day of pupation initiated adult development spontaneously within 70 days. The implantation of day 0 pupal brains into these isolated abdomens accelerated the initiation of adult development and elicited synchronous adult development. The hemolymph ecdysteroid titer of those isolated abdomens receiving implants of brains increased within 5 days and reached a maximum level of 1.5 micrograms/ml. The analysis of hemolymph ecdysteroids by reverse-phase HPLC revealed that ecdysone was the major moiety and that the ecdysteroid composition was similar to that of normal, intact pupae that had just initiated adult development. These results demonstrate that the PGs are not requisite for adult development. An increased hemolymph ecdysteroid titer was also observed in isolated abdomens from which the testes were removed and in abdomens devoid of their digestive tract. Indeed, in the latter case, the ecdysteroid titer attained much higher levels than those observed for abdomens with intact guts. Despite numerous attempts to identify the tissue(s) in the isolated abdomens responsible for the increase in ecdysteroid titer, its identity remains unknown.

Animals↗
PubMed1995

Use of juvenile hormone esterase as a novel reporter enzyme in the baculovirus expression system.

Juvenile hormone esterase (JHE) has a number of characteristics favorable for use as a reporter enzyme. It is extremely stable under a variety of adverse conditions including in organic solvent. JHE is easily detected by a rapid and sensitive colorimetric assay, and detection is facilitated by export of the enzyme from the cell. Its use is illustrated in the baculovirus expression system by promoter studies and evaluation of culture conditions necessary for optimal production of recombinant proteins. From this, it was found that protein yields were greater for Autographa californica nuclear polyhedrosis virus (AcNPV) expression vectors using the basic protein promoter to drive production of JHE than for the p10 protein or polyhedrin promoters. This has significant implications for current baculovirus expression methodologies. The effect of multiplicity of infection on protein yield was found to be insignificant between 0.1 and 10 for expression under the basic protein promoter. Yields of JHE were about 40% higher from the cell line Tn5B1-4 ('High Five') relative to the Sf21 cell line under optimized conditions for each cell line, with maximum yields obtained at 2-3 days, and 3-5 days post-infection for the two cell lines respectively when cultured in ExCell 401 medium. The presence of fetal calf serum in the cell culture medium enhanced protein yields from both cell lines. These studies demonstrate the use of JHE as a reporter enzyme for optimizing high yields of protein from the baculovirus expression system. JHE also has a potential application as a reporter enzyme in other eukaryotic systems. The advantages and use of JHE over other reporter enzyme are discussed.

Animals↗
PubMed2006

Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family.

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MS(n) (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.

Amino Acid Sequence↗