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Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor βFTZ-F1. Selective RNA silencing of βftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in βftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted βFTZ-F1 expression. Moreover, premature βftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed βFTZ-F1 expression late in the molt as steroids decline., (© 2013 Published by Elsevier Inc.)

Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling., ((c) 2009 Elsevier Inc. All rights reserved.)

The peptidergic signaling system is an ancient cell-cell communication mechanism that is involved in numerous behavioral and physiological events in multicellular organisms. We identified two novel neuropeptides in the neuronal projections innervating the salivary glands of the black-legged tick, Ixodes scapularis (Say, 1821). Myoinhibitory peptide (MIP) and SIFamide immunoreactivities were colocalized in the protocerebral cells and their projections terminating on specific cells of salivary gland acini (types II and III). Immunoreactive substances were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis: a 1,321.6-Da peptide with the sequence typical for MIP (ASDWNRLSGMWamide) and a 1,395.7-Da SIFamide (AYRKPPFNGSIFamide), which are highly conserved among arthropods. Genes encoding these peptides were identified in the available Ixodes genome and expressed sequence tag (EST) database. In addition, the cDNA encoding the MIP prepropeptide was isolated by rapid amplification of cDNA ends (RACE). In this report, we describe the anatomical structure of specific central neurons innervating salivary gland acini and identify different neuropeptides and their precursors expressed by these neurons. Our data provide evidence for neural control of salivary gland by MIP and SIFamide from the synganglion, thus leading a basis for functional studies of these two distinct classes of neuropeptides.

The domestic silkworm, Bombyx mori represents an insect model of great scientific and economic importance. Besides the establishment of a stable germline transformation using the PiggyBac vector, technically feasible methods for in vivo gene delivery and transient gene expression were developed using viral based vectors, especially Sindbis viruses and baculoviruses. The recombinant baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), commonly used for large-scale protein production in permissive cell lines or insects, has been used for foreign gene transfer into specific peptidergic cells of B. mori in vivo. Since targeted gene expression is essential for functional analysis of neuropeptide genes and their receptors, the baculovirus-mediated gene transfer can serve as a reliable approach in reverse genetic studies in the silkworm. We review various strategies employing the baculovirus vector system for transient expression of molecular markers and transcription factors in specific peptidergic cells to investigate their roles in B. mori. We also use this system for functional analysis of neuropeptide signaling in the ecdysis behavioral sequence. Our data indicate that the AcMNPV vector is suitable for efficient delivery of foreign genes and their expression directed into specific peptidergic neurons and endocrine cells of B. mori larvae and pupae. However, some modifications of the vector and steps for optimization are necessary to minimize negative effects of viral infection on the host development. The transient gene expression using the AcMNPV and other virus vectors are promising tools for analysis of molecular mechanisms underlying various neuroendocrine processes during development of B. mori.

Neuropeptides are crucial regulators of development and various physiological functions but little is known about their identity, expression and function in vectors of pathogens causing serious diseases, such as ticks. Therefore, we have used antibodies against multiple insect and crustacean neuropeptides to reveal the presence of these bioactive molecules in peptidergic neurons and cells of the ixodid tick Rhipicephalus appendiculatus. These antibodies have detected 15 different immunoreactive compounds expressed in specific central and peripheral neurons associated with the synganglion. Most central neurons arborize in distinct areas of the neuropile or the putative neurohaemal periganglionic sheath of the synganglion. Several large identified neurons in the synganglion project multiple processes through peripheral nerves to form elaborate axonal arborizations on the surface of salivary glands or to terminate in the lateral segmental organs (LSO). Additional neuropeptide immunoreactivity has been observed in intrinsic secretory cells of the LSO. We have also identified two novel clusters of peripheral neurons embedded in the cheliceral and paraspiracular nerves. These neurons project branching axons into the synganglion and into the periphery. Our study has thus revealed a complex network of central and peripheral peptidergic neurons, putative neurohaemal and neuromodulatory structures and endocrine cells in the tick comparable with those found in insect and crustacean neuroendocrine systems. Strong specific staining with a large variety of antibodies also indicates that the tick nervous system and adjacent secretory organs are rich sources of diverse neuropeptides related to those identified in insects, crustaceans or even vertebrates.

Cloning-based approach combining homology search in the Bombyx genome sequence and Rapid Amplification of cDNA Ends (RACE) resulted in annotation of 23 neuropeptide genes and different splicing variants of three genes. In total 37 neuropeptide genes in addition to bombyxin gene family have been identified in Bombyx. Comparison of available insect neuropeptide genes revealed that the silkworm genome contains most conserved neuropeptide genes except those encoding proctolin, vasopressin-like peptide and neuropeptide-like precursor 2. In addition, we identified several paralogous neuropeptide genes which have not been found in other insects. The Bombyx genome contains a triplet of paralogous genes encoding adipokinetic hormones (AKH), two genes encoding different neuropeptide Fs (NPFs) and a tandem of related SIFamide and IMFamide genes. A novel gene coding for CCHamide was cloned and its expression in the CNS and midgut was demonstrated. Differential splicing was observed for the first time in transcripts for diuretic hormones and cardioacceleratory peptides 2b. Most paralogous genes or splicing variants of the same gene showed different expression patterns in the central nervous system (CNS). These results suggest that unique duplication and differential expression of several neuropeptide genes occurred during the evolution in Bombyx. This may be an effective mechanism for functional diversification of conserved neuropeptides.

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research.

During posteclosion, insects undergo sequential processes of wing expansion and cuticle tanning. Bursicon, a highly conserved neurohormone implicated in regulation of these processes, was characterized recently as a heterodimeric cystine knot protein in Drosophila melanogaster. Here we report the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the moth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit-specific protein labeling of burs and pburs as well as crustacean cardioactive peptide in neurons of the ventral nervous system were observed in pharate larval, pupal, and adult stages. A subset of bursicon neurons located in thoracic ganglia of larvae expresses ecdysis-triggering hormone (ETH) receptors, suggesting that they are direct targets of ETH. Projections of bursicon neurons within the CNS and to neurohemal secretory sites are consistent with both central signaling and circulatory hormone functions. Intrinsic cells of the corpora cardiaca contain pburs transcripts and pburs-like immunoreactivity, whereas burs transcripts and burs-like immunoreactivity were absent in these cells. Recombinant bursicon induces both wing expansion and tanning, whereas synthetic eclosion hormone induces only wing expansion., ((c) 2007 Wiley-Liss, Inc.)

Insect ecdysis sequence is composed of pre-ecdysis, ecdysis and post-ecdysis behaviors controlled by a complex cascade of peptide hormones from endocrine Inka cells and neuropeptides in the central nervous system (CNS). Inka cells produce pre-ecdysis and ecdysis triggering hormones (ETH) which activate the ecdysis sequence through receptor-mediated actions on specific neurons in the CNS. Multiple experimental approaches have been used to determine mechanisms of ETH expression and release from Inka cells and its action on the CNS of moths and flies. During the preparatory phase 1-2 days prior to ecdysis, high ecdysteroid levels induce expression of ETH receptors in the CNS and increased ETH production in Inka cells, which coincides with expression of nuclear ecdysone receptor (EcR) and transcription factor cryptocephal (CRC). However, high ecdysteroid levels prevent ETH release from Inka cells. Acquisition of Inka cell competence to release ETH requires decline of ecdysteroid levels and beta-FTZ-F1 expression few hours prior to ecdysis. The behavioral phase is initiated by ETH secretion into the hemolymph, which is controlled by two brain neuropeptides-corazonin and eclosion hormone (EH). Corazonin acts on its receptor in Inka cells to elicit low level ETH secretion and initiation of pre-ecdysis, while EH induces cGMP-mediated ETH depletion and consequent activation of ecdysis. The activation of both behaviors is accomplished by ETH action on central neurons expressing ETH receptors A and B (ETHR-A and B). These neurons produce numerous excitatory or inhibitory neuropeptides which initiate or terminate different phases of the ecdysis sequence. Our data indicate that insect ecdysis is a very complex process characterized by two principal steps: (1) ecdysteroid-induced expression of receptors and transcription factors in the CNS and Inka cells. (2) Release and interaction of Inka cell peptide hormones and multiple central neuropeptides to control consecutive phases of the ecdysis sequence.

Structurally related ion transport peptides (ITP) and crustacean hyperglycemic hormones (CHH) are increasingly implicated in diverse metabolic and developmental functions in arthropods. We identified a conserved ITP gene encoding two peptides by alternative splicing in Manduca sexta, Bombyx mori, and Aedes aegypti: A C-terminally amidated ITP and a C-terminally unblocked ITP-like peptide (ITPL), which share common N-terminal sequences but have divergent C-termini. In the moth M. sexta, these peptides are expressed in two, regionally distinct neuronal populations in the central and peripheral nervous systems (CNS, PNS). MasITP expression is confined to the brain in five pairs of lateral neurosecretory cells (type Ia(2)) projecting ipsilateral axons into the retrocerebral complex and three to five pairs of adjacent small neurons that arborize extensively within the brain. Expression of MasITPL is comparatively weak in the brain but strong in the ventral ganglia and the PNS, where MasITP is absent. MasITPL occurs in bilaterally paired neurons of all thoracic and abdominal ganglia. In the PNS, MasITPL is coexpressed with crustacean cardioactive peptide in type II link nerve neurons (L1) of abdominal segments 2-7, which project axons into neurohemal transverse nerves. During metamorphosis, additional expression of MasITPL is observed in two pairs of small lateral neurons in the brain and one pair of ventromedial neurons in each of AG2-6. A similar pattern of differential ITP and ITPL expression was observed in the CNS and PNS of B. mori and Schistocerca americana. These distinctive cellular expression patterns suggest that ITP and ITPL have evolved specialized physiological functions in arthropods., ((c) 2006 Wiley-Liss, Inc.)

At the end of each developmental stage, insects perform the ecdysis sequence, an innate behavior necessary for shedding the old cuticle. Ecdysis triggering hormones (ETHs) initiate these behaviors through direct actions on the CNS. Here, we identify the ETH receptor (ETHR) gene in the moth Manduca sexta, which encodes two subtypes of GPCR (ETHR-A and ETHR-B). Expression of ETHRs in the CNS coincides precisely with acquisition of CNS sensitivity to ETHs and behavioral competence. ETHR-A occurs in diverse networks of neurons, producing both excitatory and inhibitory neuropeptides, which appear to be downstream signals for behavior regulation. These peptides include allatostatins, crustacean cardioactive peptide (CCAP), calcitonin-like diuretic hormone, CRF-like diuretic hormones (DHs) 41 and 30, eclosion hormone, kinins, myoinhibitory peptides (MIPs), neuropeptide F, and short neuropeptide F. In particular, cells L(3,4) in abdominal ganglia coexpress kinins, DH41, and DH30, which together elicit the fictive preecdysis rhythm. Neurons IN704 in abdominal ganglia coexpress CCAP and MIPs, whose joint actions initiate the ecdysis motor program. ETHR-A also is expressed in brain ventromedial cells, whose release of EH increases excitability in CCAP/MIP neurons. These findings provide insights into how innate, centrally patterned behaviors can be orchestrated via recruitment of peptide cotransmitter neurons.

Background: At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system., Results: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence., Conclusions: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

In insects, steroid hormones named ecdysteroids elicit molting and metamorphosis. The prothoracic gland (PG) is a predominant source of ecdysteroids, where their biosynthesis (ecdysteroidogenesis) is regulated by several neuropeptides. Here, we report that FMRFamide-related peptides (FaRPs) regulate ecdysteroidogenesis through direct innervation of the PG in the silkworm Bombyx mori. We purified a previously uncharacterized Bombyx FaRP, DPSFIRFamide, and identified the corresponding Bombyx FMRFamide gene (Bommo-FMRFamide, BRFa), which encodes three additional FaRPs. All BRFa peptides suppressed ecdysteroidogenesis in the PG by reducing cAMP production by means of the receptor for Bommo-myosuppressin, another FaRP we have previously shown to act as a prothoracicostatic factor. BRFa is predominantly expressed in neurosecretory cells of thoracic ganglia, and the neurons in the prothoracic ganglion innervate the PG to supply all four peptides to the gland surface. Electrophysiological recordings during development confirmed the increased firing activity of BRFa neurons in stages with low PG activity and decreased ecdysteroid levels in the hemolymph. To our knowledge, this study provides the first report of peptides controlling ecdysteroidogenesis by direct innervation.

Corazonin is a highly conserved neuropeptide hormone of wide-spread occurrence in insects yet is associated with no universally recognized function. After discovery of the corazonin receptor in Drosophila, we identified its ortholog in the moth, Manduca sexta, as a prelude to physiological studies. The corazonin receptor cDNA in M. sexta encodes a protein of 436 amino acids with seven putative transmembrane domains and shares common ancestry with its Drosophila counterpart. The receptor exhibits high sensitivity and selectivity for corazonin when expressed in Xenopus oocytes (EC(50) approximately 200 pM) or Chinese hamster ovary cells (EC(50) approximately 75 pM). Northern blot analysis locates the receptor in peripheral endocrine Inka cells, the source of preecdysis- and ecdysis-triggering hormones. Injection of corazonin into pharate larvae elicits release of these peptides from Inka cells, which induce precocious preecdysis and ecdysis behaviors. In vitro exposure of isolated Inka cells to corazonin (25-100 pM) induces preecdysis- and ecdysis-triggering hormone secretion. Using corazonin receptor as a biosensor, we show that corazonin concentrations in the hemolymph 20 min before natural preecdysis onset range from 20 to 80 pM and then decline over the next 30-40 min. These findings support the role of corazonin signaling in initiation of the ecdysis behavioral sequence. We propose a model for peptide-mediated interactions between Inka cells and the CNS underlying this process in insect development.

Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, we found Inka cells in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviours in pharate larvae of Bombyx, indicating functional similarity of these peptides. We isolated several PETH-immunoreactive peptides from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and identified the gene encoding two putative ETHs in the mosquito Anopheles gambiae. Inka cells also are stained with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, our enzyme immunoassays show that these antisera cross-react with PETH and ETH. Our results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioural sequence.

Inka cells of the epitracheal endocrine system produce peptide hormones involved in the regulation of insect ecdysis. In the silkworm Bombyx mori, injection of Inka cell extract into pharate larvae, pupae or adults activates the ecdysis behavioural sequence. In the present study, we report the identification of three peptides in these extracts, pre-ecdysis-triggering hormone (PETH), ecdysis-triggering hormone (ETH) and ETH-associated peptide (ETH-AP), which are encoded by the same cDNA precursor. Strong immunoreactivity associated with each peptide in Inka cells prior to ecdysis disappears during each ecdysis, indicating complete release of these peptides. Injection of either PETH or ETH alone is sufficient to elicit the entire ecdysis behavioural sequence through the direct action on abdominal ganglia; cephalic and thoracic ganglia are not required for the transition from pre-ecdysis to ecdysis behaviour. Our in vitro data provide evidence that these peptides control the entire ecdysis behavioural sequence through activation of specific circuits in the nervous system. Ecdysis of intact larvae is associated with the central release of eclosion hormone (EH) and elevation of cyclic 3',5'-guanosine monophosphate (cGMP) in the ventral nerve cord. However, injection of ETH into isolated abdomens induces cGMP elevation and ecdysis behaviour without a detectable release of EH, suggesting that an additional central factor(s) may be involved in the activation of this process. Our findings provide the first detailed account of the natural and hormonally induced behavioural sequence preceding larval, pupal and adult ecdyses of B. mori and highlight significant differences in the neuro-endocrine activation of pre-ecdysis and ecdysis behaviours compared with the related moth, Manduca sexta.

Initiation of the ecdysis behavioural sequence in insects requires activation of the central nervous system (CNS) by pre-ecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which are released from the Inka cells of the epitracheal glands. Here, we show that the developmental events preceding larval and pupal ecdysis of Manduca sexta involve a dual action of ecdysteroids on the epitracheal glands and CNS. The low steroid levels in freshly ecdysed and feeding larvae are associated with small-sized epitracheal glands, reduced peptide production in Inka cells and insensitivity of the CNS to ETH. The elevated ecdysteroid levels before each ecdysis lead to a dramatic enlargement of Inka cells and increased production of peptide hormones and their precursors. As blood ecdysteroids reach peak levels, the CNS becomes responsive to Inka cell peptides. These effects of natural ecdysteroid pulses can be experimentally induced by injection of 20-hydroxyecdysone or the ecdysteroid agonist tebufenozide (RH-5992) into ecdysed larvae, thus stimulating peptide production in Inka cells and inducing CNS sensitivity to ETH. A direct steroid action on the CNS is demonstrated by subsequent treatment of isolated nerve cords from ecdysed larvae with 20-hydroxyecdysone and ETH, which results in pre-ecdysis or ecdysis bursts. Our data show that ecdysteroid-induced transcriptional activity in both the epitracheal glands and the CNS are necessary events for the initiation of the ecdysis behavioural sequence.

Insects shed their old cuticle by performing the ecdysis behavioural sequence. To activate each subunit of this set of programmed behaviours in Manduca sexta, specific central ganglia are targeted by pre-ecdysis-triggering (PETH) and ecdysis-triggering (ETH) hormones secreted from Inka cells. PETH and ETH act on each abdominal ganglion to initiate, within a few minutes, pre-ecdysis I and II, respectively. Shortly thereafter, ETH targets the tritocerebrum and suboesophageal ganglion to activate the ecdysis neural network in abdominal ganglia through the elevation of cyclic GMP (cGMP) levels. However, the onset of ecdysis behaviour is delayed by inhibitory factor(s) from the cephalic and thoracic ganglia. The switch from pre-ecdysis to ecdysis is controlled by an independent clock in each abdominal ganglion and is considerably accelerated after removal of the head and thorax. Eclosion hormone (EH) appears to be one of the central signals inducing elevation of cGMP levels and ecdysis, but these actions are quite variable and usually restricted to anterior ganglia. EH treatment of desheathed ganglia also elicits strong production of cGMP in intact ganglia, suggesting that this induction occurs via the release of additional downstream factors. Our data suggest that the initiation of pre-ecdysis and the transition to ecdysis are regulated by stimulatory and inhibitory factors released within the central nervous system after the initial actions of PETH and ETH.

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence.

Ecdysis behavior in the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) is triggered through reciprocal peptide signaling between the central nervous system and the epitracheal endocrine system. Recent evidence indicates that eclosion hormone may initiate endocrine events leading to ecdysis through its action on epitracheal glands to cause the release of ecdysis-triggering hormone (ETH). Here, we report that direct exposure of epitracheal glands to eclosion hormone in vitro leads to secretion of ETH. The threshold concentration of eclosion hormone needed to evoke release of ETH is approximately 3 pmol l-1. Eclosion hormone also induces elevation of cyclic GMP, but not cAMP, concentration in epitracheal glands at concentrations similar to those causing release of ETH. Both cGMP and 8-Br-cGMP mimic the secretory action of eclosion hormone. The sensitivity of the secretory response to eclosion hormone occurs during a narrow window of development, beginning approximately 8 h prior to pupal ecdysis. However, eclosion hormone can cause elevation of cGMP levels in epitracheal glands long before they acquire competence to release ETH, showing that the initial portion of the signal transduction cascade is in place early in development, but that the absence of a downstream step in the cascade prevents secretion. Measurements of cGMP levels in epitracheal glands during the ecdysis sequence show a sudden elevation some 30 min after the onset of pre-ecdysis, well after ETH secretion has been initiated. ETH secretion can therefore be viewed as a two-step process, beginning at pre-ecdysis when cGMP levels are relatively low, followed by a massive release resulting from a logarithmic elevation of cGMP levels.

Parasitism of Manduca sexta (Lepidoptera: Sphingidae) larvae by the braconid wasp Cotesia congregata (Hymenoptera: Braconidae) leads to accumulation of peptides in host neurons and neurosecretory cells of the central nervous system (CNS) and neurons and endocrine/paracrine cells of the midgut. This accumulation has now facilitated the characterization of two new members of the FLRFamide family from midguts of parasitized larvae. The peptides, given the names F24 and F39, are 24 and 39 amino acids in length with the sequences VRDYPQLLDSGMKRQDVVHSFLRFamide and YAEAAGEQVPEYQALVRDYPQLLDSGMKRQDVVHSFLRFamide. The sequence of F24 is identical to the C-terminal 24 amino acids of F39. The C-terminal 10-mer of each is identical to a previously characterized decapeptide neurohormone (F10). This sequence is preceded by a potential processing site. In nonparasitized insects F39 was present at several-fold the amount of F24. In parasitized insects F24 and F39 accumulate in the middle and posterior regions of the midgut, which are enriched in endocrine/paracrine cells reacting with FLRFamide antisera. In the combined brain and subesophageal ganglion F39 was not detected and the amount of F24 never exceeded 2 fmol per Br/SEG. Of the three peptides, only F10 was found in the hemolymph. Thus, F24 and F39 may be intermediates in the biosynthesis of F10 and may themselves be released locally from endocrine/paracrine cells in the midgut epithelium.

Ecdysis, the shedding of cuticle at the end of each life stage, is critical to the postembryonic development of insects. The endocrine regulation of ecdysis has been highlighted by the recent description of the epitracheal endocrine system in the tobacco hornworm Manduca sexta, which produces ecdysis-triggering hormone (Mas-ETH). This peptide hormone initiates pre-ecdysis and ecdysis through a direct action on the central nervous system. Here we show that ETH-immunoreactivity and ecdysis-triggering activity in epitracheal glands of the silkworm Bombyx mori are attributable to a 23 amino acid peptide, Bom-ETH. The complete amino acid sequence of Bom-ETH is SNEAFDEDVMGYVIKSNKNIPRM-NH2. Synthetic Bom-ETH was prepared and shown to be chemically and biologically identical to the native substance. Injection of Bom-ETH leads to pre-ecdysis and ecdysis in B. mori pharate larvae and pupae as well as comparable stages of M. sexta. Exposure of the isolated nervous system to Bom-ETH triggers pre-ecdysis and ecdysis burst patterns corresponding to the natural behavior. Bom-ETH belongs to an extended family of multifunctional neurohormones and hormones found in arthropods and molluscs.

Lymphadenitis with persisting fistualas in children is almost always conditioned by specific inflammation, tuberculosis, zoonoses, actinomycoses. Tuberculosis of the lymph nodes can either be primary or post-primary. In this case report we have dealt with a case of a rare primary lymph node tuberculosis that arose as a result of vaccination complications. Disturbance of cellular immunity was the reason for BCG lymphadenitis. In references, the reasons for BCG-itis as well as vaccination contra-indications have been described.

The association of SLE with tuberculosis (TB) was studied in a group of 388 patients with SLE monitored between 1953-1994. TB was diagnosed in 14 patients (3.6%). The occurrence of septic fevers in SLE patients that did not respond to glucocorticoid therapy indicated the possibility of complication with TB. SLE-associated TB included miliary and far-advanced pulmonary and extrapulmonary forms. Three patients from our group died due to myco-bacterial infection and one patient died of active SLE and TB. The treatment was successful in nine patients. Early diagnosis and appropriate management are mandatory in SLE associated TB, which otherwise may have a potentially fatal outcome.

Developing insects repeatedly shed their cuticle by means of a stereotyped behavior called ecdysis, thought to be initiated by the brain peptide eclosion hormone. Here an ecdysis-triggering hormone, Mas-ETH, is described from the tobacco hornworm Manduca sexta. Mas-ETH contains 26 amino acids and is produced by a segmentally distributed endocrine system of epitracheal glands (EGs). The EGs undergo a marked reduction in volume, appearance, and immunohistochemical staining during ecdysis, at which time Mas-ETH is found in the hemolymph. Injection of EGs extract or synthetic Mas-ETH into pharate larvae, pupae, or adults initiates preecdysis within 2 to 10 minutes, followed by ecdysis. Sensitivity to injected Mas-ETH appears much earlier before ecdysis and occurs with shorter latency than that reported for eclosion hormone. The isolated central nervous system responds to Mas-ETH, but not to eclosion hormone, with patterned motor bursting corresponding to in vivo preecdysis and ecdysis. Mas-ETH may be an immediate blood-borne trigger for ecdysis through a direct action on the nervous system.

An unusual cause of obstructive sleep apnea in a boy with hemophilia B who was urgently intubated during the night because of suspected bleeding into the airway is analysed. The cause of airway obstruction was a floating papilloma hanging from false cord. At inspirium the tumor was moving immediately above the vocal cords. This was manifested during sleep by noisy snoring and numerous apneic pauses. When the child was awake he had no respiratory problems. After the tumor was removed, the boy breathed freely during sleep. However, the papillomas recur in various parts of the larynx and repeated surgical treatment by factor IX replacement: therapy is necessary.

Manduca sexta larvae that are parasitized by the braconid wasp Cotesia congregata enter a state of developmental arrest following emergence of the wasp larvae from the host. These fifth instar hosts linger for 2 to 3 weeks without resuming feeding, molting, or metamorphosis once the wasps emerge. Immunohistochemical staining with antiserum against FMRFamide revealed dramatic accumulation of FMRFamide-like peptide(s) in the gut nervous and endocrine systems of the developmentally arrested larvae when compared to that observed in unparasitized feeding or starved larvae. Specifically, the number of immunopositive cells and the intensity of staining was enhanced in the neurons of the frontal ganglion, the axons and axon terminals on the midgut surface, and in the gastric endocrine cells. These results were confirmed using ELISA to show that the relative amounts of FMRFamide-like peptides in midgut extracts were highly elevated in the parasitized larvae relative to the fed or starved unparasitized larvae. These data suggest that FMRFamide-like peptides in developmentally arrested larvae are produced in a significantly larger number of gastric endocrine cells, and that the rate of release of the peptides may be suppressed, or the rate of their synthesis may be elevated. Localization of FMRFamide-like peptides in the gastric endocrine cells of C. congregata is also described.

Fifth instar larvae of Manduca sexta that were parasitized by the braconid wasp Cotesia congregata failed to develop after the parasitoid larvae emerged, and these host larvae lingered for 2-3 weeks in a quiescent, nonfeeding state without initiating a larval molt or metamorphosis. This study was focused on the neuroendocrine changes associated with the host's developmental arrest. Immunohistochemical studies suggested that the host brain neurosecretory cells as well as their axon terminals in the corpora cardiaca-corpora allata complex accumulated multiple neuropeptides. The extent of accumulation in cells and axons increased with time, so that hosts examined 7-14 days after the wasps emerged showed the most intense staining with antibodies against prothoracicotropic hormone, bombyxin, allatotropin, allatostatin, diuretic hormone, eclosion hormone, proctolin, and FMRFamide. Increased levels of prothoracicotropic hormone and FMRFamide-like peptides in the brains of parasitized larvae were confirmed using Western blots and enzyme-linked immunosorbent assay (ELISA), respectively. Starvation of the unparasitized larvae induced some accumulation of the neuropeptides; however, the intensity of staining and number of immunopositive cells and axons were in most cases clearly higher in the parasitized larvae. Our results suggest that accumulation of the neuropeptides is associated with developmental arrest of parasitized larvae. Because a similar developmental arrest occurs in a wide range of parasitized insects, our findings may have relevance for many other species. Moreover, these data illustrate the potential value of using parasitized M. sexta larvae as a model for studying the mechanisms governing the rates of neuropeptide expression, processing, packaging, and release, as well as providing a rich source of neuropeptides, thus facilitating their isolation and characterization.

Antibodies generated against bombyxin and prothoracicotropic hormone (PTTH) of Bombyx mori and allatotropin, allatostatin, and diuretic hormone (DH) of Manduca sexta react with distinct sets of cells in the central nervous system of Drosophila larvae, pupae, and adults. Brain neurons immunoreactive with antibodies to bombyxin, PTTH, and DH are in strikingly similar positions to their lepidopteran counterparts, indicating that at least some Drosophila neuroendocrine cells are homologous to those of lepidopterans. Allatotropin and allatostatin-immunopositive neurons of Drosophila differ from those of lepidopterans, but many of them are identical with neurons that express the FMRFamide gene. Antibodies to bombyxin, PTTH, allatostatin, and DH also stain axons and axon terminals in the neurohemal part of the ring gland, and all tested antibodies except that against bombyxin show positive reaction in the neurohemal area of the ventral ganglion. Although immunoreactivity with all antibodies is variable during development, the highest levels of staining are found at developmental stages when the neuropeptides would be expected to be functioning. A genetic analysis of neuropeptide expression and function has been initiated by analyzing immunoreactivity in mutants that have prolonged larval life associated with imaginal disc overgrowth. Two of these mutants, dlg and dco, show abnormally strong immunoreactivity for allatotropin during the extended larval period and the former also show increased staining with the PTTH antibody. The reduced ecdysteroid titer and delayed or blocked metamorphosis in the mutants may be a result of altered neuropeptide production, which is probably secondary to the imaginal disc overgrowth.

In the course of the last three decades a gradually supplemented group of 627 patients was subjected to prognostic analysis, incl. 388 with systemic lupus erythematosus, 160 with diffuse scleroderma, 51 with dermato- and polymyositis and 28 with a mixed connective tissue syndrome. Taking into account the onset of the disease, sex, extent of organ affection and therapy as well as the survival period and mortality, the conclusions of the statistical evaluation revealed that more than 90% of patients survived for more than 5 years: 92.2% with systemic lupus erythematosus, 94.3% with diffuse scleroderma 93.3% with dermato- and polymyositis and 100% with mixed connective tissue syndrome; a ten-year survival was recorded in cca 80% patients (77.6% with systemic lupus erythematosus, 81.1% with diffuse scleroderma, 89.7% with dermato- and polymyositis); a 15-year survival was recorded still by more than 50% patients (53.4% with systemic lupus erythematosus, 58.2% with diffuse scleroderma and as many as 80.0% with dermato- and polymyositis). The above data indicate considerable improvement of the prospects of these diseases, whereby the more than treble prolongation of survival of patients as compared with the previous period is due to effective, although not causal immunotherapy, in particular in systemic lupus erythematosus and dermato- and polymyositis.

The influence of thymosterin on immunological reactivity of NZB mice was investigated. Thymosterin was administered three times per week for a period of six weeks to male and female NZB mice. Only lymphocytes from untreated female mice gave the positive results in the macrophage migration inhibition test using native DNA as the stimulating antigen. This effect was suppressed in thymosterin treated females. The administration of thymosterin therefore appears to correct the in vitro delayed hypersensitivity to DNA antigen.

In our article is referred to the recognition of collagenosis in the demarcated whole district with 100,000 inhabitants. The occurrence of these diseases is lower than the occurrence of other rheumatic diseases. Our results as well as the observations of other authors confirm that the lupus erythematodes and the diffuse sclerodermia are not rare diseases even in our circumstances and with their evolution tendency they shorten the lives of the affected persons. The high recognition coefficient with large probability corresponds to the real incidence of these two diseases, which can be achieved only by an observation of many years and repeated controls of the correctness of the diagnosis.

Prolonged repeated analyses of sera from 98 patients with systemic lupus erythematosus (SLE) and 76 patients with similar diffuse diseases of connective tissue by a radioisotope membrane binding method for the assessment of antibodies against double-stranded ribonucleic acid (anti-ds-RNA) revealed that these antibodies are most frequently encountered in patients with the evolutive form of SLE (53%), but also in approximately one fifth of patients with non- evolutive SLE and in patients with similar diffuse diseases of connective tissue. Anti-ds-RNA are not linked with stages of clinical activity; their occurrence is on the whole independent on the occurence of antibodies against ds-DNA and nuclear ribonucleoprotein. There is a very close association with the incidence of antibodies against denatured DNA and a substantially small association with antibodies against acid nuclear antigen (Sm). The occurrence of anti-ds-RNA is a favourable prognostic sign, as in the majority of patients who had such antibodies at any time in their serum, later the disease can be suppressed by treatment, while patients who never had anti-ds-RNA in serum remain for a long time in the active stage of the disease.

A family with hereditary C2 deficiency was discovered in Czechoslovakia. The proband is a 47-year-old female with a SLE-like syndrome and zero activity of the classical complement pathway. Functional CH50, C1, C2, and C4 estimations for all family members revealed a homozygous C2 deficiency in both the proband and her elder sister, and several heterozygotic C2-deficient individuals. The defect segregates with haplo-type HLA A25, B18, DR2.

Data are presented demonstrating the prevention of spontaneous autoimmune disease in NZB/Swiss mice treated natural double-stranded RNA. The successful treatment of animals was followed both by improvement in clinical manifestations and by the migration inhibition test performed with DNA as antigen. The reasons for the successful employment of natural double-stranded RNA are thought to be in its physicochemical properties.