The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect moulting hormone.

The developmental events occurring during moulting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the moulting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20E (20-hydroxyecdysone), are mediated by four cytochrome P450 (P450) enzymes, encoded by genes in the Halloween family. Orthologues of the Drosophila Halloween genes phantom (phm; CYP306A1), disembodied (dib; CYP302A1), shadow (sad; CYP315A1) and shade (shd; CYP314A1) were obtained from the endocrinological model insect, the tobacco hornworm Manduca sexta. Expression of these genes was studied and compared with changes in the ecdysteroid titre that controls transition from the larval to pupal stage. phm, dib and sad, which encode P450s that mediate the final hydroxylations in the biosynthesis of ecdysone, were selectively expressed in the prothoracic gland, the primary source of ecdysone during larval and pupal development. Changes in their expression correlate with the haemolymph ecdysteroid titre during the fifth (final) larval instar. Shd, the 20-hydroxylase, which converts ecdysone into the more active 20E, is expressed in tissues peripheral to the prothoracic glands during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The results indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses triggering moulting.

Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca(2+)- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis.

The synthesis of ecdysteroids by the lepidopteran prothoracic gland is regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). In Manduca sexta glands, PTTH stimulates several events including Ca(2+) influx, Ca(2+)-dependent cAMP generation and the activation of several protein kinases. In the present study, the path by which PTTH stimulates extracellular signal-activated regulated kinase (ERK) phosphorylation was investigated using PTTH and second messenger analogs. The results indicate that Ca(2+)-dependent processes, other than cAMP generation, play the major role in PTTH stimulation of ERK phosphorylation in larval prothoracic glands, that cAMP-dependent events increase in importance during later development and that PTTH-stimulated ERK phosphorylation is highest in larval glands. The decline in PTTH-stimulated ERK phosphorylation associated with metamorphosis results from decreased ERK levels and an increased basal rate of ERK phosphorylation. The data suggest that the role or importance of components of the PTTH signal transduction cascade are not fixed and can change during development.

Activation of an extracellular signal-regulated kinase (ERK) by the insect prothoracicotropic hormone.

Ecdysteroid hormones are crucial in controlling the growth, molting and metamorphosis of insects. The predominant source of ecdysteroids in pre-adult insects is the prothoracic gland, which is under the acute control of the neuropeptide hormone prothoracicotropic hormone (PTTH). Previous studies using the tobacco hornworm, Manduca sexta, have shown that PTTH stimulates ecdysteroid synthesis via a series of events, including the activation of protein kinase A and the 70 kDa S6 kinase (p70(S6k)). In this study, PTTH was shown to stimulate also mitogen-activated protein kinase (MAPK) phosphorylation and activity in the Manduca prothoracic gland. The MAPK involved appears to be an extracellular signal-regulated kinase (ERK) homologue. The ERK phosphorylation inhibitors PD 98059 and UO 126 blocked basal and PTTH-stimulated ERK phosphorylation and ecdysteroid synthesis. PTTH-stimulated ERK activity may be important for both rapid regulation of ecdysteroid synthesis and for longer-term changes in the size and function of prothoracic gland cells.

Dynamic regulation of prothoracic gland ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain extracts have identical effects.

Multiple assays were conducted in order to determine if the recently available recombinant prothoracicotropic hormone (rPTTH) from Manduca sexta is identical, or similar, to the natural hormone and if results from its use in a variety of assays confirm, or are inconsistent with, previous studies over the past 20years on PTTH action using brain extract. Brain extracts and rPTTH showed similar, if not identical, effects on the cell biology of Manduca prothoracic gland cells with the following results: increased levels of cAMP (adenosine 3':5' cyclic monophosphate) synthesis; requirement for extracellular Ca(2+) in in vitro studies; ecdysteroidogenesis stimulation in vitro; stimulation of general and specific protein synthesis; immunocytochemical identification of the two lateral cells in each brain hemisphere as the source of PTTH (the prothoracicotropes); the ability of antibodies to rPTTH to inhibit ecdysteroidogenesis stimulation in vitro; and the multiple phosphorylation of the ribosomal protein S6. The data revealed that brain extract and rPTTH show equivalent effects in all of the assays, indicating that this rPTTH is the natural PTTH of Manduca and that the data generated with brain extracts over the past two decades are indeed relevant.

cDNA cloning and expression of a hormone-regulated heat shock protein (hsc 70) from the prothoracic gland of Manduca sexta.

The brain neuropeptide prothoracicotropic hormone (PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein (hsc 70). To further understand the possible roles of hsc 70, hsc 70 cDNA clones were isolated from a tobacco hornworm (Manduca sexta) prothoracic gland cDNA library. All sequenced clones were highly homologous to the Drosophila hsc 70-4 isoform. Manduca hsc 70 mRNA levels during the last larval instar exhibited a peak at the onset of wandering and a peak that coincided with the major pre-metamorphic peak of ecdysteroid synthesis. Manipulations of the glands' hormonal milieu showed that hsc 70 mRNA levels respond to 20-hydroxyecdysone, dibutyryl cAMP, PTTH and the JH analogue hydroprene. The protein and mRNA data suggest that hsc 70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex.

Cloning of a beta1 tubulin cDNA from an insect endocrine gland: developmental and hormone-induced changes in mRNA expression.

A rapid increase in ecdysteroid hormone synthesis results when the insect prothoracic gland is stimulated with prothoracicotropic hormone (PTTH), a brain neuropeptide hormone. PTTH also stimulates the specific synthesis of several proteins, one of which is a beta tubulin. To further understand the possible roles of beta tubulin in the prothoracic gland, beta tubulin cDNA clones were isolated from a tobacco hornworm (Manduca sexta) gland cDNA library. Sequence analysis indicated that these clones were assignable to the beta1 tubulin isoform. Gland beta1 tubulin mRNA levels during the last larval instar and early pupal-adult development exhibited peaks that coincided with peaks in ecdysteroid synthesis. Manipulations of the glands hormonal milieu showed that beta1 tubulin mRNA levels respond to 20 hydroxyecdysone and PTTH. The data also support our earlier proposal that the prothoracic gland beta1 tubulin gene is ubiquitously expressed but exhibits tissue- and developmental-specific regulation of transcription and translation.

Control of ecdysteroidogenesis: activation and inhibition of prothoracic gland activity.

The ecdysteroid hormones, mainly 20-hydroxyecdysone (20E), play a pivotal role in insect development by controlling gene expression involved in molting and metamorphosis. In the model insect Manduca sexta the production of ecdysteroids by the prothoracic gland is acutely controlled by a brain neurohormone, prothoracicotropic hormone (PTTH). PTTH initiates a cascade of events that progresses from the influx of Ca2+ and cAMP generation through phosphorylation of the ribosomal protein S6 and S6-dependent protein synthesis, and concludes with an increase in the synthesis and export of ecdysteroids from the gland. Recent studies indicate that S6 phosphorylation probably controls the steroidogenic effect of PTTH by gating the translation of selected mRNAs whose protein products are required for increased ecdysteroid synthesis. Inhibition of S6 phosphorylation prevents an increase in PTTH-stimulated protein synthesis and subsequent ecdysteroid synthesis. Two of the proteins whose translations are specifically stimulated by PTTH have been identified, one being a beta tubulin and the other a heat shock protein 70 family member. Current data suggest that these two proteins could be involved in supporting microtubule-dependent protein synthesis and ecdysone receptor assembly and/or function. Recent data also indicate that the 20E produced by the prothoracic gland feeds back upon the gland by increasing expression and phosphorylation of a specific USP isoform that is a constituent of the functional ecdysone receptor. Changes in the concentration and composition of the ecdysone receptor complex of the prothoracic gland could modulate the gland's potential for ecdysteroid synthesis (e.g. feedback inhibition) by controlling the levels of enzymes or other proteins in the ecdysteroid biosynthetic pathway.

Bombyx and Manduca prothoracicotropic hormones: an immunologic test for relatedness.

Prothoracicotropic hormone (PTTH) controls the synthesis of ecdysteroids (molting hormones) by the insect prothoracic gland and thereby plays a critical role in insect growth, molting, and metamorphosis. PTTH has been purified, and cDNA clones obtained, from only one insect, the silkmoth Bombyx mon. Recently, a partial amino acid sequence was obtained for-a putative PTTH from the tobacco hornworm, Manduca sexta, and the data suggested an unexpected homology to a vertebrate retinoid binding protein rather than to the Bombyx PTTH. In this study, a monoclonal antibody against the Bombyx PTTH was added to in vitro assays that assess the ability of partially purified Manduca PTTH to stimulate prothoracic gland ecdysteroid and protein synthesis. The results indicate that Bombyx and Manduca PTTHs are indeed members of the same protein family that have likely diverged in the PTTH receptor-binding region.

Prothoracicotropic hormone-regulated expression of a hsp 70 cognate protein in the insect prothoracic gland.

In Manduca sexta, ecdysteroids coordinate molting and metamorphosis of insects and are produced by the prothoracic glands under the acute control of the brain neuropeptide prothoracicotropic hormone (PTTH). PTTH stimulates rapid ecdysteroidogenesis accompanied by specific increases in the synthesis and accumulation of three proteins, including one with M(r) = 70 kDa. This 70-kDa protein is a constitutively expressed member of the heat shock protein 70 family (hsc 70). Levels of this hsc 70 vary in a prothoracic gland-specific manner during development as does its PTTH-stimulated synthesis when assayed in vitro. The accumulation of hsc 70 may be regulated by abrupt changes in its turnover rate. The PTTH-stimulated increase in hsc 70 synthesis is dependent upon both translational and transcriptional events. Hsc 70 expression in the prothoracic gland may be required for changes in gland growth, e.g., protein content, that underlie alterations in ecdysteroid production.

Stereospecific, mechanism-based, suicide inhibition of a cytochrome P450 involved in ecdysteroid biosynthesis in the prothoracic glands of Manduca sexta.

The first required step in ecdysteroid (molting hormone) biosynthesis, dietary cholesterol (C) conversion to 7-dehydrocholesterol (7dC) via 7,8-dehydrogenation, is mediated by a microsomal cytochrome-P450 monooxygenase specific to the larval prothoracic gland. A subsequent series of unknown "black-box" oxidations of 7dC result in the unusual ring geometry (cis-A/B) and functionality (6-keto-7-ene-14-alpha-ol) of the ecdysteroids and has been thought to involve the initial formation of alpha-5,6-epoxy-7-dehydrocholesterol (alpha epo7dC). Pharmacological studies indicated that conversion of C to 7dC in prothoracic gland homogenates was strongly and equally inhibited by the isomeric cholesterol substrate analogues alpha- and beta-5,6-epoxycholesterol (alpha- and beta epoC) and alpha- and beta-5,6-iminocholesterol (alpha- and beta iminoC). With respect to the conversion of C to ecdysteroids by disrupted glands, however, the two alpha-isomeric substrates were 10-fold more inhibitory than were their beta-analogues. Indeed, alpha amino C was as active as the non-specific pyrimidyl cytochrome-P450 monooxygenase inhibitor fenarimol that shows moderate toxicity in many insect species. All four cholesterol analogues competitively inhibited cholesterol 7,8-dehydrogenation, but only alpha epoC and possibly alpha iminoC were desaturated to delta 7-products. Although the KmS (and KiS) for all the substrates were similar (1.7-6.0 x 10(-5) M), the Vmax for alpha epoC dehydrogenation was eight-fold higher than that of C, making it a superior substrate for following this reaction in ecdysteroidogenic tissues rich in endogenous C. The 7,8-dehydrogenation of alpha epoC and alpha iminoC by prothoracic glands would produce the potentially reactive intermediates, alpha epo7dC and alpha imino7dC, respectively. They, in turn, could then undergo facile, acid-catalyzed ring-opening to the allylic-stabilized carbo-cation electrophiles. These very reactive, transient species, if formed in the active site of the monooxygenase, would then alkylate either the heme group or the apoprotein of the cytochrome or both, leading to the irreversible inhibition of the enzyme. The present data show that alpha epoC and probably alpha iminoC are mechanism-based suicide inhibitors of the enzyme catalyzing cholesterol 7,8-dehydrogenation and may be the prototypes of a new class of selective insect control agents.

Prothoracicotropic hormone elicits a rapid, developmentally specific synthesis of beta tubulin in an insect endocrine gland.

The production of ecdysteroid (molting) hormones by the insect prothoracic gland is controlled primarily by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). PTTH stimulates also the specific synthesis of three proteins in the prothoracic glands of the tobacco hornworm Manduca sexta (Rybczynski and Gilbert, 1994). Here, one of these proteins, p50 is identified as a beta tubulin. The ability of PTTH to stimulate beta tubulin synthesis increased dramatically late on Day 3 of the 10-day fifth larval instar. At this time and later, beta tubulin synthesis in response to PTTH in vitro could be detected in some prothoracic glands 5-10 min after the onset of stimulation, and newly synthesized beta tubulin entered the microtubule pool within 12 min. Levels of beta tubulin in the glands of fifth instar larvae, measured by immunoblot, changed in a tissue-specific manner that paralleled or presaged circulating ecdysteroid levels. The accumulation of beta tubulin in PTTH-stimulated prothoracic glands resulted from increased transcription and translation and not from a decreased protein turnover rate. Pulse-chase experiments indicate that the newly synthesized beta tubulin had a very short half-life in vitro (approximately 0.5 hr). Studies with cycloheximide and actinomycin D indicated that beta tubulin synthesis and ecdysteroid synthesis are coregulated and that beta tubulin synthesis is regulated in a unique manner relative to most other prothoracic gland proteins. We conclude that beta tubulin levels may play an important role in ecdysteroidogenesis, perhaps by influencing the dynamics of microtubule-dependent secretion or interorganelle movement of ecdysteroid precursors.

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.

Ecdysteroid regulation of olfactory protein expression in the developing antenna of the tobacco hawk moth, Manduca sexta.

During adult metamorphosis, the moth olfactory neurons and their glia-like support cells pass through a coordinated and synchronous development. By 60% of development, the olfactory system is anatomically complete, but functional maturation does not occur until about 90% of development. Maturation is characterized by the onset of odorant sensitivity in the sensory neurons and the expression of certain antennal-specific proteins including odorant binding proteins (OBPs) and odorant degrading enzymes (ODEs). The OBPs have been cloned and sequenced, and are thus useful models for investigating the molecular mechanisms coordinating final maturation of the developing olfactory system. The ecdysteroid hormones have been observed to regulate many cellular level neuronal changes during adult metamorphosis. In particular, the late pupal decline in ecdysteroids is known to influence programmed death of nerves and muscles at the end of metamorphosis. Experiments are presented here which indicate that this decline in ecdysteroids also induces the expression of the OBPs. Normal OBP expression occurs 35-40 h before adult emergence. In culture, OBP expression could be induced at least 90 h before adult emergence by the premature removal of ecdysteroid. This premature expression was blocked by culturing tissue in the presence of the biologically active ecdysteroid 20-hydroxyecdysone. These findings suggest that maturation of the olfactory system is regulated by the decline in ecdysteroids, and support the view that olfactory development, in general, may be coordinated by changing levels of pupal ecdysteroids.

Molecular cloning and sequencing of general odorant-binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: comparisons with other insect OBPs and their signal peptides.

Odorant-binding proteins (OBPs) are small, water-soluble proteins uniquely expressed in olfactory tissue of insects and vertebrates. OBPs are present in the aqueous fluid surrounding olfactory sensory dendrites and are thought to aid in the capture and transport of hydrophobic odorants into and through this fluid. OBPs may represent the initial biochemical recognition step in olfaction, because they transport odorants to the receptor neurons. Insect OBPs are represented by multiple classes: pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBP1 and GOBP2). PBPs associate with pheromone-sensitive neurons, while GOBPs associate with general odorant-sensitive neurons. Analysis of N-terminal amino acid sequences of 14 insect OBPs isolated from six species indicated that the PBPs were variable and the GOBPs were highly conserved. However, inferred properties of these proteins were based only on partial sequence data. We now report the full-length sequences of a GOBP1 and GOBP2 from the moth Manduca sexta and compare these sequences with those of PBPs from three species, including M. sexta, Antheraea polyphemus, and A. pernyi. We also compare these with a GOBP2 of A. pernyi, previously identified only as a novel OBP. These comparisons fully support our N-terminal analysis. The signal peptide sequences of seven insect OBPs reveal conserved sequences within OBP classes, but not between OBP classes even within the same animal species. This suggests that multiple OBPs may be coexpressed in the same cell type, but differentially processed in a class-specific manner. Properties of the GOBPs suggest that general olfaction is broadly receptive at the periphery. Properties of the PBPs suggest that pheromone olfaction is discriminatory at the periphery, and that the initial biochemical steps in pheromone detection may play an active role in odor perception.

Antennal-specific pheromone-degrading aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori.

Female moths produce blends of odorant chemicals, called pheromones. These precise chemical mixtures both attract males and elicit appropriate mating behaviors. To locate females, male moths must rapidly detect changes in environmental pheromone concentration. Therefore, the regulation of pheromone concentration within antennae, their chief organ of smell, is important. We describe antennal-specific aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori that are capable of catabolizing long chain, unsaturated aldehydes such as their aldehyde pheromones. These soluble enzymes are associated uniquely with male and female antennae and have molecular masses of 175 and 130 kDa, respectively. The A. polyphemus aldehyde oxidase has been localized to the olfactory sensilla which contain the pheromone receptor cell dendrites. These same sensilla contain a previously described sensilla-specific esterase that degrades the acetate ester component of A. polyphemus pheromone. We propose that sensillar pheromone-degrading enzymes modulate pheromone concentration in the receptor space and hence play a dynamic role in the pheromone-mediated reproductive behaviors of these animals.

A pheromone-degrading aldehyde oxidase in the antennae of the moth Manduca sexta.

Antennae of the tobacco hornworm moths Manduca sexta contain an aldehyde oxidase (AOX) that oxidizes aldehydes to carboxylic acids. The enzyme, which is distinguishable from aldehyde-oxidizing activities in other tissues, is secreted into the receptor lymph that bathes the primary olfactory dendrites. First detectable about 3 d before eclosion, AOX levels increase through the first day after eclosion. This parallels the development of the antennal responsiveness to bombykal (a male attractant aldehydic pheromone produced by female M. sexta) and trans-2-hexenal (an aldehyde commonly found in leaves). The AOX is about 60% more abundant in antennae of males than in antennae of females. The antennal AOX is a dimer with Mr of 295 kDa and is capable of oxidizing a variety of aldehydes. Of all aldehydes examined, the pheromone bombykal was the best substrate with an apparent Km of 5 microM, whereas the next best substrate, benzaldehyde, had an apparent Km of 255 microM. Using kinetic parameters estimated in vitro and the assumption of first-order kinetics, the half-life of bombykal in sensilla was estimated to be about 0.6 msec. The affinity of the antennal AOX for bombykal, its location in the receptor lymph, and its pattern of developmental expression all suggest that it plays a role in modulating the sensitivity of adult M. sexta to aldehyde odors and, in particular, the sensitivity of males to the pheromone bombykal.

Mitochondrial protein synthesis in interspecific somatic cell hybrids.

The fusion of an oligomycin (OLI)-resistant mutant of mouse LM(TK-) cells to a chloramphenicol (CAP)-resistant mutant of AK412 Chinese hamster cells resulted in a series of interspecific somatic cell hybrids. Hybrids selected in HAT medium retained only mouse mitochondrial genomes while hybrids selected in HAT plus CAP and OLI retained both hamster and mouse mitochondrial genomes in approximately equal amounts. Nuclear-coded mitochondrial proteins from both parental species were incorporated into mitochondria in all of the hybrids. However, the mitochondrially coded proteins of three individually isolated hybrid cell lines were predominantly mouse-specific, with only trace amounts of hamster protein detected.

Soluble and particulate forms of rat catechol-O-methyltransferase distinguished by gel electrophoresis and immune fixation.

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.