The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Expression of stress-related genes in the parthenogenetic forms of the pea aphid, Acyrthosiphon pisum.

Aphids are an economically important group of insects that have an intricate life cycle with seasonal polyphenism. This study aimed to explore the physiological background of aphid migration from unfavorable nutritional conditions to a new, intact host plant. Specifically, the relative expression of stress/metabolism-related genes and changes in metabolic reserves were determined for the winged and wingless forms of female pea aphids, Acyrthosiphon pisum, under two different nutritional conditions. The expression level was determined for the following sets of genes: the adipokinetic hormone (AKH) and its receptor, enzymes involved in carbohydrate and lipid metabolism, detoxifying enzymes, and genes encoding exoskeleton/cuticular proteins and cytoskeleton proteins. In both forms, the transcription of the adipokinetic hormone was upregulated during nutritional stress, whereas its receptor mRNA levels remained unchanged. Similarly, the expression of genes engaged in glycogen and triglyceride degradation was elevated. Glycogen reserves and phospholipids appeared to be used during stress. In comparison, nutrient rich reproductively active females of both forms appeared to use triglycerides. Moreover, we revealed changes in the mRNA level of the detoxifying genes delta-class glutathione S-transferase (GST-δ) and cytochrome P450 monooxygenase (CYP450), as well as the CP gene (which encodes exoskeleton/cuticular proteins) and the cofilin gene (the products of which influence cytoskeleton organization). These results indicate the possible correlation between nutritional stress, energy content, AKH, and the stress-related enzymes of different metabolic pathways in winged and wingless forms of A. pisum.

Modulatory effects of bombyxin on ecdysteroidogenesis in Bombyx mori prothoracic glands.

In the present study, we investigated the modulatory effects of ecdysteroidogenesis of prothoracic glands (PGs) by bombyxin, an endogenous insulin-like peptide in the silkworm, Bombyx mori. The results showed that bombyxin stimulated ecdysteroidogenesis during a long-term incubation period and in a dose-dependent manner. Moreover, the injection of bombyxin into day 4-last instar larvae increased ecdysteroidogenesis 24h after the injection, indicating its possible in vivo function. Phosphorylation of the insulin receptor and Akt, and the target of rapamycin (TOR) signaling were stimulated by bombyxin, and stimulation of Akt phosphorylation and TOR signaling appeared to be dependent on phosphatidylinositol 3-kinase (PI3K). Bombyxin inhibited the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK), and the inhibition appeared to be PI3K-independent. Bombyxin-stimulated ecdysteroidogenesis was blocked by either an inhibitor of PI3K (LY294002) or a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, AICAR), indicating involvement of the PI3K/Akt and AMPK signaling pathway. Bombyxin did not stimulate extracellular signal-regulated kinase (ERK) signaling of PGs. Bombyxin, but not prothoracicotropic hormone (PTTH) stimulated cell viability of PGs. In addition, bombyxin treatment also affected mRNA expression levels of insulin receptor, Akt, AMPKα, -ß, and -γ in time-dependent manners. These results suggest that bombyxin modulates ecdysteroidogenesis in B. mori PGs during development.

Un primer análisis de la investigación en México sobre los determinantes sociales de la salud: 2005-2012 / A first analysis of research on social determinants of health in Mexico: 2005-2012

Objetivo. Examinar la investigación hecha en México sobre los determinantes sociales de la salud (DSS) durante el periodo 2005-2012 con base en la caracterización del sistema nacional de investigación en salud y la producción científica sobre este tema. Material y métodos. Análisis en dos etapas: revisión documental de fuentes oficiales sobre investigación en salud en México y búsqueda sistemática de literatura sobre DSS. Resultados. Los DSS fueron mencionados en el Programa de Acción Específico de Investigación en Salud 2007-2012, pero no figuran en las estrategias y objetivos; en su lugar, se enfatizan primordialmente aspectos de infraestructura y administrativos. En el periodo se publicaron 145 artículos sobre DSS, cuyas temáticas más abordadas fueron "condiciones de salud", "sistemas de salud" y "nutrición y obesidad". Conclusiones. A pesar de que existe investigación en México sobre DSS, la instrumentación de esos hallazgos en políticas de salud no se ha implementado. El Programa Sectorial de Salud 2013-2018 representa una ventana de oportunidad para posicionar resultados de investigación que promuevan políticas de equidad en salud.

Objective. To examine the research on social determinants of health (SDH) produced in Mexico during the period 2005-2012, based on the characterization of the national health research system and the scientific production on this topic. Materials and methods. Two-stage analyses: Review of Mexican documents and official sources on health research and systematic bibliographic review of the literature on SDH. Results. Although SDH were mentioned in the Specific Action Plan for Health Research 2007-2012, they are not implemented in strategies and goals, as the emphasis is put mostly in infrastructure and administrative aspects of research. In the period studied, 145 articles were published on SDH topics such as health conditions, health systems and nutrition and obesity. Conclusions. In spite of the availability of research on SDH in Mexico, the operationalization of such findings into health policies has not been possible. The current Sectorial Program on Health 2013-2018 represents a window of opportunity to position research findings that promote health equity policies.

In search for non-steroidogenic functions of the prothoracic glands of the desert locust, Schistocerca gregaria: a peptidomic and proteomic approach.

The only well established function of the prothoracic glands (PGs) of insects is the production of ecdysteroids. In gregarious locusts, like in most insect species, the PGs degenerate soon after the adult molt. In this way they resemble the thymus of mammals, a gland with an important role in the build up of the immune system in young animals. In adult solitarious locusts the PGs persist much longer, however without producing substantial amounts of ecdysteroids. In the literature the existence of a well developed rough endoplasmic reticulum and Golgi complex system has been repeatedly reported, suggesting an active role in peptide or/and protein synthesis and release. The nature of the secreted products remained unknown. Our pepdidomic analysis of an acidified methanolic extract of PGs of last instar gregarious nymphs did not yield any indication for the presence of known locust or other peptides. The peptide release assay was also negative. For our proteomic analysis, we developed an EST-based identification strategy. We successfully identified 50 protein spots on a two dimensional map. In addition to typical protein synthesis-related proteins, a number of proteins with a role in detoxification processes were found, suggesting some role of the PGs in the defense system.

An FXPRLamide neuropeptide induces seasonal reproductive polyphenism underlying a life-history tradeoff in the tussock moth.

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH(2) (FXPRLa) neuropeptides: DH, PBAN, and α-, ß-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.

Transcriptional regulation of the gene for prothoracicotropic hormone in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) is one of key players in regulation of insect growth, molting, metamorphosis, diapause, and is expressed specifically in the two pairs of lateral PTTH-producing neurosecretory cells in the brain. Analysis of cis-regulatory elements of the PTTH promoter might elucidate the regulatory mechanism controlling PTTH expression. In this study, the PTTH gene promoter of Bombyx mori (Bom-PTTH) was cloned and sequenced. The cis-regulatory elements in Bom-PTTH gene promoter were predicted using Matinspector software, including myocyte-specific enhancer factor 2, pre-B-cell leukemia homeobox 1, TATA box, etc. Transient transfection assays using a series of fragments linked to the luciferase reporter gene indicated that the fragment spanning -110 to +33 bp of the Bom-PTTH promoter showed high ability to support reporter gene expression, but the region of +34 to +192 bp and -512 to -111 bp repressed the promoter activity in the BmN and Bm5 cell lines. Electrophoretic mobility shift assays demonstrated that the nuclear protein could specifically bind to the region spanning -124 to -6 bp of the Bom-PTTH promoter. Furthermore, we observed that the nuclear protein could specifically bind to the -59 to -30 bp region of the Bom-PTTH promoter. A classical TATA box, TATATAA, localized at positions -47 to -41 bp, which is a potential site for interaction with TATA box binding protein (TBP). Mutation of this TATA box resulted in no distinct binding band. Taken together, TATA box was involved in regulation of PTTH gene expression in B. mori.

Biochemical, molecular, and functional characterization of PISCF-allatostatin, a regulator of juvenile hormone biosynthesis in the mosquito Aedes aegypti.

Aedes aegypti PISCF-allatostatin or allatostatin-C (Ae-AS-C) was isolated using a combination of high performance liquid chromatography and enzyme-linked immunosorbent assay (ELISA). The matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrum of positive ELISA fractions revealed a molecular mass of 1919.0 Da, in agreement with the sequence qIRYRQCYFNPISCF, with bridged cysteines. This sequence was confirmed by matrix-assisted laser desorption/ionization tandem TOF/TOF mass spectrometry analysis. The corresponding Ae-AS-C cDNA was amplified by PCR, and the sequence of the peptide was confirmed. An in vitro radiochemical assay was used to study the inhibitory effect of synthetic Ae-AS-C on juvenile hormone biosynthesis by the isolated corpora allata (CA) of adult female A. aegypti. The inhibitory action of synthetic Ae-AS-C was dose-dependent; with a maximum at 10(-9) m. Ae-AS-C showed no inhibitory activity in the presence of farnesoic acid, an immediate precursor of juvenile hormone, indicating that the Ae-AS-C target is located before the formation of farnesoic acid in the pathway. The sensitivity of the CA to inhibition by Ae-AS-C in the in vitro assay varied during the adult life; the CA was most sensitive during periods of low synthetic activity. In addition, the levels of Ae-AS-C in the brain were studied using ELISA and reached a maximum at 3 days after eclosion. These studies suggest that Ae-AS-C is an important regulator of CA activity in A. aegypti.

Effects of starvation and mating on corpora allata activity and allatotropin (Manse-AT) gene expression in Manduca sexta.

The levels of three alternatively spliced mRNAs from the Manduca sexta allatotropin (Manse-AT) gene were determined following physiological manipulations during the larval, pupal and adult stages; starvation of larvae, induction of pupal diapause and adult mating experience. The juvenile hormone biosynthetic activity of the corpora allata (CA) was also determined in starved larvae and in mated and unmated females. Starvation of early fifth instar larvae specifically increased the amount of one Manse-AT mRNA that is predicted to encode Manse-AT and two related peptides, Manse-ATL-I and -II. The normal rapid decrease in the activity of the CA in last instar larvae was not observed in starved insects which maintained a relatively high rate of JH biosynthesis for at least 3 days. Diapause induction resulted in a small increase in one Manse-AT mRNA, but levels were much lower compared to those observed in larvae or adults. During the first 4 days of adult life, Manse-AT mRNA levels were not changed as a result of mating. However, in mated females, the rate of JH biosynthesis gradually increased, in sharp contrast to the relatively low level of CA activity seen in virgin females. These observations suggest the elevated activity of the CA in mated females is not simply due to the increased level of Manse-AT mRNA.

Neuroparsin transcripts as molecular markers in the process of desert locust (Schistocerca gregaria) phase transition.

Desert locust swarms occasionally cause severe ecological and economic damage, particularly in countries of northwest Africa. However, the physiological mechanisms underlying locust phase transition, the switch of the solitarious to the gregarious phase, remain elusive. Therefore, identification of molecular changes linked to this phenomenon represents a primary requirement to start unraveling this enigma. The present paper provides novel information on phase-related molecular markers for locust phase transition. We present a detailed quantitative real-time RT-PCR analysis of two distinct neuroparsin precursor transcripts (Scg-NPP3 and Scg-NPP4) in the brain and in abdominal tissues of gregarious and solitarious desert locusts (Schistocerca gregaria). Our data reveal different temporal changes of these transcripts in the fat body during the adult stage of both phases. We, hereby, present novel scientific evidence for a phase-dependent regulation of these particular peptide hormone encoding transcripts and assign them as possible molecular markers in the process of locust phase transition.

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.

Identification of a novel prothoracicostatic hormone and its receptor in the silkworm Bombyx mori.

The insect brain regulates the activity of the prothoracic glands to secrete ecdysteroids, which affect growth, molting, and metamorphosis. Here we report the identification of a novel prothoracicostatic factor and its receptor in the silkworm Bombyx mori. The prothoracicostatic factor purified from pupal brains of B. mori is a decapeptide with the conserved structure of an insect myosuppressin and thus named Bommo-myosuppressin. Bommo-myosuppressin dose dependently suppressed the cAMP level and inhibited ecdysteroidogenesis in the larval prothoracic glands at much lower concentrations than the prothoracicostatic peptide, the other prothoracicostatic factor reported previously. In vitro analyses using a prothoracic gland incubation method revealed that Bommo-myosuppressin and prothoracicostatic peptide regulate the prothoracic gland activity via different receptors. In situ hybridization and immunohistochemistry revealed the existence of Bommo-myosuppressin in the brain neurosecretory cells projecting to neurohemal organs in which it is stored. We also identified and functionally characterized a specific receptor for Bommo-myosuppressin and showed its high expression in the prothoracic glands. All these results suggest that Bommo-myosuppressin functions as a prothoracicostatic hormone and plays an important role in controlling insect development.

The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of ELAV protein.

Although the Drosophila melanogaster erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa EWG protein in neurons, while protein expression elsewhere or of other isoforms is below detection at this stage. This posttranscriptional control is important, as broad expression of EWG can be lethal. In this paper, we show that ELAV, a neuron-specific RNA binding protein, is necessary to regulate EWG protein expression in ELAV-null eye imaginal disc clones and that ELAV is sufficient for EWG expression in wing disc imaginal tissue after ectopic expression. Further, analysis of EWG expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in ELAV-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and ELAV-deficient eye imaginal discs and in wild-type and ectopic ELAV-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to ELAV levels. The data presented in this paper are consistent with a mechanism in which ELAV increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism in which stability of specific splice forms is enhanced by ELAV. Additionally, we report that ELAV promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. ELAV, however, is not involved in all neuron-enriched splice events.

Identification of a prothoracicostatic peptide in the larval brain of the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) stimulates ecdysteroid biosynthesis in the prothoracic gland (PG) of insects. A peptide inhibiting ecdysteroid biosynthesis in the PG was isolated from the extracts of 2,000 larval brains of the silkworm, Bombyx mori, using a protocol that included four reversed-phase high performance liquid chromatography procedures. The primary structure of this prothoracicostatic peptide (Bom-PTSP) was determined to be H-Ala-Trp-Gln-Asp-Leu-Asn-Ser-Ala-Trp-NH(2). This neuropeptide has the same sequence as Mas-MIP-I, a myoinhibitory peptide previously isolated from the ventral nerve cord of the tobacco hornworm, Manduca sexta, and is highly homologous with the N-terminal portion of vertebrate peptides of the galanin family. This peptide inhibited PTTH-stimulated ecdysteroidogenesis in the PG at both the spinning and feeding stages, which indicates that Bom-PTSP interferes with PTTH-stimulated ecdysteroidogenesis.

Expression of the Manduca sexta allatotropin gene in cells of the central and enteric nervous systems.

Manduca sexta allatotropin (Mas-AT) was isolated and first characterized as a peptide that stimulated juvenile hormone biosynthesis in adult lepidopteran corpora allata and was subsequently shown to have cardioacceleratory activity in the pharate adult. In this study, we identified the cells in the nervous system of the insect that contain mRNA encoding Mas-AT and immunoreactivity against a polyclonal antiserum to Mas-AT. In larvae, Mas-AT mRNA and immunoreactivity was most abundant in two cells in the frontal ganglion, which project their axons down the recurrent nerve toward the gut, and in cells in the terminal abdominal ganglion. Lower levels of Mas-AT mRNA were detected in the brain and subesophageal ganglion. In the pupal and pharate adult stages, we detected Mas-AT mRNA and immunoreactivity in cells of the abdominal ganglia and in additional cells in the terminal abdominal ganglion. These additional cells in the ventral nerve cord that express Mas-AT during the pupal and pharate adult stages include cells that differentiate during metamorphosis as well as cells that exist in larvae but do not begin to express Mas-AT until these later developmental stages. Some of the cells that exhibit Mas-AT immunoreactivity lack Mas-AT mRNA, suggesting that the antisera used in this and previous studies recognizes other peptides in addition to Mas-AT. This pattern of expression suggests that Mas-AT may mediate multiple physiological functions during the life cycle of the insect, including the larval stage in which no function has yet been described for the peptide.

Angiotensin II and angiotensin-converting enzyme as candidate compounds modulating the effects of testis ecdysiotropin in testes of the gypsy moth, Lymantria dispar1.

Lymantria dispar testes synthesize immunodetectable ecdysteroid in vitro in response to the brain peptide, testis ecdysiotropin (TE), acting primarily via a cascade involving Gi protein, diacyl glycerol, and phosphokinase C. However, a component of TE activation also involves the opposite cascade, Gs protein, cAMP, and phosphokinase A. Excess cAMP inhibits the action of TE, acting as a feedback modulator. Here, we show that bovine angiotensin II (AII) and bovine angiotensin converting enzyme (ACE) act like cAMP, inducing synthesis of immunodetectable ecdysteroid by pupal testes in vitro, but are antagonistic to coincubated TE. In addition, an insect ACE antibody clearly stains the spermatogenic cells through all stages of development, as well as testis sheath tissue where ecdysteroid is synthesized. AII induces synthesis of cAMP by pupal testes in vitro. Therefore, insect homologs of mammalian AII and ACE are good candidates for the peptides responsible for the cAMP cascade and as modulators of TE action in lepidopteran testes. Saralasin, an analog of AII that blocks angiotensin receptors in mammals, behaved like AII in inducing ecdysteroid secretion with ecdysteroidogenic effects additive to either angiotensin or ACE. Therefore, the receptors for the insect form of angiotensin on lepidopteran testis cells are probably different from those in mammals. Saralasin also inhibited ecdysteroid synthesis when combined with TE, as did AII.

Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland.

The diazepam-binding inhibitor (DBI) is a 10-kDa highly evolutionarily conserved multifunctional protein. In mammals, one of DBI's functions is in the activation of steroid hormone biosynthesis via binding to a specific outer mitochondrial membrane receptor (benzodiazepine receptor, BZD) and promoting cholesterol transport to the inner membrane. In this work, a multitiered approach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothoracic glands (PGs). First, both DBI protein and messenger RNA (mRNA) levels were correlated with peak PG ecdysteroid production. In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27 and inhibited anti-DBI antibodies. The DBI protein was found distributed throughout PG cells, including regions of dense mitochondria, supposed subcellular sites of ecdysteroid synthesis. Finally, a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaffinity labeling. These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cholesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.

Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway.

A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward arrow-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels.

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.