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Background: Multimodal breast cancer treatment may cause side effects reflected in patient-reported outcomes and/or symptom scores at the time of treatment planning for adjuvant radiotherapy. In our department, all patients have been assessed with the Edmonton Symptom Assessment System (ESAS; a questionnaire addressing 11 major symptoms and wellbeing on a numeric scale of 0-10) at the time of treatment planning since 2016. In this study, we analyzed ESAS symptom severity before locoregional radiotherapy., Patients and Methods: Retrospective review of 132 patients treated between 2016 and 2021 (all comers in breast-conserving or post-mastectomy settings, different radiotherapy fractionations) was performed. All ESAS items and the ESAS point sum were analyzed to identify subgroups with higher symptom burden and thus need for additional care measures., Results: The biggest patient-reported issues were fatigue, pain, and sleep problems. Patients with triple negative breast cancer reported a higher symptom burden (mean 30 versus 20, p = 0.038). Patients assigned to adjuvant endocrine therapy had the lowest point sum (mean 18), followed by those on Her-2-targeting agents without chemotherapy (mean 19), those on chemotherapy with or without other drugs (mean 26), and those without systemic therapy (mean 41), p = 0.007. Those with pathologic complete response after neoadjuvant treatment had significantly lower anxiety scores (mean 0.7 versus 1.8, p = 0.03) and a trend towards lower depression scores, p = 0.09., Conclusion: Different surgical strategies, age, and body mass index did not impact on ESAS scores, while the type of adjuvant systemic therapy did. The effect of previous neoadjuvant treatment and unfavorable tumor biology (triple negative) emerged as important factors associated with symptom burden, albeit in different domains. ESAS data may facilitate identification of patients who should be considered for additional supportive measures to alleviate specific symptoms., (© 2024. The Author(s).)

Intravascular processing of triglyceride-rich lipoproteins (TRLs) is crucial for delivery of dietary lipids fueling energy metabolism in heart and skeletal muscle and for storage in white adipose tissue. During the last decade, mechanisms underlying focal lipolytic processing of TRLs along the luminal surface of capillaries have been clarified by fresh insights into the functions of lipoprotein lipase (LPL); LPL's dedicated transporter protein, glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1); and its endogenous inhibitors, angiopoietin-like (ANGPTL) proteins 3, 4, and 8. Key discoveries in LPL biology include solving the crystal structure of LPL, showing LPL is catalytically active as a monomer rather than as a homodimer, and that the borderline stability of LPL's hydrolase domain is crucial for the regulation of LPL activity. Another key discovery was understanding how ANGPTL4 regulates LPL activity. The binding of ANGPTL4 to LPL sequences adjacent to the catalytic cavity triggers cooperative and sequential unfolding of LPL's hydrolase domain resulting in irreversible collapse of the catalytic cavity and loss of LPL activity. Recent studies have highlighted the importance of the ANGPTL3-ANGPTL8 complex for endocrine regulation of LPL activity in oxidative organs (e.g., heart, skeletal muscle, brown adipose tissue), but the molecular mechanisms have not been fully defined. New insights have also been gained into LPL-GPIHBP1 interactions and how GPIHBP1 moves LPL to its site of action in the capillary lumen. GPIHBP1 is an atypical member of the LU (Ly6/uPAR) domain protein superfamily, containing an intrinsically disordered and highly acidic N-terminal extension and a disulfide bond-rich three-fingered LU domain. Both the disordered acidic domain and the folded LU domain are crucial for the stability and transport of LPL, and for modulating its susceptibility to ANGPTL4-mediated unfolding. This review focuses on recent advances in the biology and biochemistry of crucial proteins for intravascular lipolysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kristensen, Leth-Espensen, Kumari, Grønnemose, Lund-Winther, Young and Ploug.)

Background/aim: Oncological care has faced several challenges during the COVID-19 pandemic, e.g. treatment delay and worsening symptoms. Patient-reported anxiety, depression and sleep quality might have changed due to these special circumstances. Therefore, we analyzed the symptom burden of patients treated with palliative radiotherapy at our center., Patients and Methods: A retrospective study was performed of 50 consecutive patients and the results were compared to those obtained in a previous pre-COVID study. The Edmonton Symptom Assessment Scale was employed to assess the preradiotherapy symptoms., Results: The highest mean scores were reported for pain in activity (3.2) and dry mouth (3.1). Regarding anxiety, sadness/depression and sleep, the corresponding scores were 1.5, 1.2 and 2.7, respectively. Compared to the previous study, no significant increases were found. Most items had numerically lower mean values, e.g. anxiety (1.5 vs. 2.7). Both study populations had comparable median age (70.5 vs. 70 years), gender distribution and proportion of patients with bone metastases. However, there were two significant imbalances, namely a lower proportion of patients with prostate cancer (12 vs. 30%, p=0.02) and breast cancer (0 vs. 12%, p=0.02)., Conclusion: In patients who showed up for radiation treatment planning, the suspected increase in anxiety, sadness/depression and sleep disturbance was not demonstrable. It is not known whether or not patients with substantial worries chose to decline referral to palliative radiotherapy. Therefore, comprehensive large-scale studies of patterns of care are needed to fully understand the impact of COVID-19-related measures., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (T m is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (T m increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca 2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL)., Competing Interests: Author Contributions Conceptualization and Design, M.M., A.G., W.D.-B., A.-M.L.W., M.B., and G.R.; Methodology, all authors; Resources, all authors; Investigation, all authors; Formal Analysis, all authors; Writing – Original Draft, W.D.-B., A.-M.L.W., M.M., M.B., and G.R.; Writing – Review & Editing, W.D.-B., A.-M.L.W., M.M., M.B., and G.R.; Data Curation, W.D.-B., A.-M.L.W., P.S., R.L.S., C.M., F.A., K.S., M.B., and G.R. Funding Acquisition, P.S., C.M., R.L.S., K.S., M.M., and G.R.; Project Administration, M.M. and G.R.; Supervision, G.R. Declaration of Interests W.D.-B. and A.-M.L.W. are CaDo Biotechnology IvS employees. The compounds studied are part of a patent application wholly owned by CaDo Biotechnology IvS, Denmark. K.S. has previously consulted for Novartis and Rigel Pharmaceuticals and currently receives grant funding from Novartis on topics unrelated to this manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Proteins containing Ly6/uPAR (LU) domains exhibit very diverse biological functions and have broad taxonomic distributions in eukaryotes. In general, they adopt a characteristic three-fingered folding topology with three long loops projecting from a disulfide-rich globular core. The majority of the members of this protein domain family contain only a single LU domain, which can be secreted, glycolipid anchored, or constitute the extracellular ligand binding domain of type-I membrane proteins. Nonetheless, a few proteins contain multiple LU domains, for example, the urokinase receptor uPAR, C4.4A, and Haldisin. In the current review, we will discuss evolutionary aspects of this protein domain family with special emphasis on variations in their consensus disulfide bond patterns. Furthermore, we will present selected cases where missense mutations in LU domain-containing proteins leads to dysfunctional proteins that are causally linked to genesis of human disease.

In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H(+)-ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H(+)-ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR). This led to identification of a series of uncommon o-hydroxybenzylated flavanones and chalcones, i.e., chamanetin (8), isochamanetin (9), isouvaretin (10), uvaretin (11), dichamanetin (12), and diuvaretin (15). Preparative-scale isolation of the active metabolites allowed determination of IC50 values for inhibition of the PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. These revealed a strong correlation between o-hydroxybenzyl substituents and PM H(+)-ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H(+)-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentration-dependent activity profile. These extracts were selected for semi-high-resolution fungal PM H(+)-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H(+)-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) led to identification of chebulagic acid (1) and tellimagrandin II (2) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC50 values for PM H(+)-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H(+)-ATPase with inhibitory effect on the growth of S. cerevisiae.

The most severe form of human malaria is caused by the parasite Plasmodium falciparum. Despite the current need, there is no effective vaccine and parasites are becoming resistant to most of the antimalarials available. Therefore, there is an urgent need to discover new drugs from targets that have not yet suffered from drug pressure with the aim of overcoming the problem of new emerging resistance. Membrane transporters, such as P. falciparum Ca(2+)-ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), have been proposed as potentially good antimalarial targets. The present investigation focuses on: (a) the large-scale purification of PfATP6 for maintenance of its enzymatic activity; (b) screening for PfATP6 inhibitors from a compound library; and (c) the selection of the best inhibitors for further tests on P. falciparum growth in vitro. We managed to heterologously express in yeast and purify an active form of PfATP6 as previously described, although in larger amounts. In addition to some classical SERCA inhibitors, a chemical library of 1680 molecules was screened. From these, we selected a pool of the 20 most potent inhibitors of PfATP6, presenting half maximal inhibitory concentration values in the range 1-9 μm. From these, eight were chosen for evaluation of their effect on P. falciparum growth in vitro, and the best compound presented a half maximal inhibitory concentration of ~ 2 μm. We verified the absence of an inhibitory effect of most of the compounds on mammalian SERCA1a, representing a potential advantage in terms of human toxicity. The present study describes a multidisciplinary approach allowing the selection of promising PfATP6-specific inhibitors with good antimalarial activity., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

In insects, many complex behaviors, including olfactory memory, are controlled by a paired brain structure, the so-called mushroom bodies (MB). In Drosophila, the development, neuroanatomy, and function of intrinsic neurons of the MB, the Kenyon cells, have been well characterized. Until now, several potential neurotransmitters or neuromodulators of Kenyon cells have been anatomically identified. However, whether these neuroactive substances of the Kenyon cells are functional has not been clarified yet. Here we show that a neuropeptide precursor gene encoding four types of short neuropeptide F (sNPF) is required in the Kenyon cells for appetitive olfactory memory. We found that activation of Kenyon cells by expressing a thermosensitive cation channel (dTrpA1) leads to a decrease in sNPF immunoreactivity in the MB lobes. Targeted expression of RNA interference against the sNPF precursor in Kenyon cells results in a highly significant knockdown of sNPF levels. This knockdown of sNPF in the Kenyon cells impairs sugar-rewarded olfactory memory. This impairment is not due to a defect in the reflexive sugar preference or odor response. Consistently, knockdown of sNPF receptors outside the MB causes deficits in appetitive memory. Altogether, these results suggest that sNPF is a functional neuromodulator released by Kenyon cells.

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

The central complex is a prominent set of midline neuropils in the insect brain, known to be a higher locomotor control center that integrates visual inputs and modulates motor outputs. It is composed of four major neuropil structures, the ellipsoid body (EB), fan-shaped body (FB), noduli (NO), and protocerebral bridge (PB). In Drosophila different types of central complex neurons have been shown to express multiple neuropeptides and neurotransmitters; however, the distribution of corresponding receptors is not known. Here, we have mapped metabotropic, G-protein-coupled receptors (GPCRs) of several neurotransmitters to neurons of the central complex. By combining immunocytochemistry with GAL4 driven green fluorescent protein, we examined the distribution patterns of six different GPCRs: two serotonin receptor subtypes (5-HT(1B) and 5-HT(7)), a dopamine receptor (DopR), the metabotropic GABA(B) receptor (GABA(B)R), the metabotropic glutamate receptor (DmGluR(A)) and a short neuropeptide F receptor (sNPFR1). Five of the six GPCRs were mapped to different neurons in the EB (sNPFR1 was not seen). Different layers of the FB express DopR, GABA(B)R, DmGluR(A,) and sNPFR1, whereas only GABA(B)R and DmGluR(A) were localized to the PB. Finally, strong expression of DopR and DmGluR(A) was detected in the NO. In most cases the distribution patterns of the GPCRs matched the expression of markers for their respective ligands. In some nonmatching regions it is likely that other types of dopamine and serotonin receptors or ionotropic GABA and glutamate receptors are expressed. Our data suggest that chemical signaling and signal modulation are diverse and highly complex in the different compartments and circuits of the Drosophila central complex. The information provided here, on receptor distribution, will be very useful for future analysis of functional circuits in the central complex, based on targeted interference with receptor expression., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Drosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophilag the regulation of DILP production and release in adult fruit flies is poorly understood. Here we investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR and NKD, in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, we show DTK-immunoreactive axon terminations close to the presumed dendrites of the IPCs, and DTKR immunolabeling in these cells. Second, we utilized targeted RNA interference to knock down expression of the DTK receptor, DTKR, in IPCs and monitored the effects on Dilp transcript levels in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. We also measured trehalose and lipid levels as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, we targeted the IPCs with RNAi or ectopic expression of the other DTK receptor, NKD, but found no effect on survival at starvation. Our results suggest that DTK signaling, via DTKR, regulates the brain IPCs.

The central complex of the insect brain is an integration center, receiving inputs from many parts of the brain. In Drosophila it has been associated with the control of both locomotor and visually correlated behaviors. The central complex can be divided into several substructures and is comprised of a large number of neuronal types. These neurons produce classical neurotransmitters, biogenic amines, and different neuropeptides. However, the distribution of neurotransmitters and neuromodulators in central-complex circuits of Drosophila is poorly known. By immunolabeling and GAL4-directed expression of marker proteins, we analyzed the distribution of acetylcholine, glutamate, GABA, monoamines, and eight different neuropeptides; Drosophila tachykinin, short neuropeptide F, myoinhibitory peptide, allatostatin A, proctolin, SIFamide, neuropeptide F, and FMRFamide. All eight neuropeptides were localized to the fan-shaped body, the largest substructure of the central complex, and were mapped to different layers within this structure. Several populations of peptide-immunoreactive tangential and columnar neurons were identified, of which some colocalized acetylcholine. Fewer peptides were found to be expressed in the other substructures: the ellipsoid body, the protocerebral bridge, and the noduli. The ellipsoid body and the protocerebral bridge were innervated by extrinsic peptide expressing neurons. Our findings reveal that numerous neuropeptides are expressed in the central complex and that each peptide has a distinct distribution pattern, suggesting important roles for neuropeptides as neuromediators and cotransmitters in this brain area., (© 2010 Wiley-Liss, Inc.)

The sarcoplasmic (SERCA 1a) Ca2+-ATPase is a membrane protein abundantly present in skeletal muscles where it functions as an indispensable component of the excitation-contraction coupling, being at the expense of ATP hydrolysis involved in Ca2+/H+ exchange with a high thermodynamic efficiency across the sarcoplasmic reticulum membrane. The transporter serves as a prototype of a whole family of cation transporters, the P-type ATPases, which in addition to Ca2+ transporting proteins count Na+, K+-ATPase and H+, K+-, proton- and heavy metal transporting ATPases as prominent members. The ability in recent years to produce and analyze at atomic (2·3-3 Å) resolution 3D-crystals of Ca2+-transport intermediates of SERCA 1a has meant a breakthrough in our understanding of the structural aspects of the transport mechanism. We describe here the detailed construction of the ATPase in terms of one membraneous and three cytosolic domains held together by a central core that mediates coupling between Ca2+-transport and ATP hydrolysis. During turnover, the pump is present in two different conformational states, E1 and E2, with a preference for the binding of Ca2+ and H+, respectively. We discuss how phosphorylated and non-phosphorylated forms of these conformational states with cytosolic, occluded or luminally exposed cation-binding sites are able to convert the chemical energy derived from ATP hydrolysis into an electrochemical gradient of Ca2+ across the sarcoplasmic reticulum membrane. In conjunction with these basic reactions which serve as a structural framework for the transport function of other P-type ATPases as well, we also review the role of the lipid phase and the regulatory and thermodynamic aspects of the transport mechanism.

Thapsigargin (Tg), a specific inhibitor of sarco/endoplasmic Ca(2+)-ATPases (SERCA), binds with high affinity to the E2 conformation of these ATPases. SERCA inhibition leads to elevated calcium levels in the cytoplasm, which in turn induces apoptosis. We present x-ray crystallographic and intrinsic fluorescence data to show how Tg and chemical analogs of the compound with modified or removed side chains bind to isolated SERCA 1a membranes. This occurs by uptake via the membrane lipid followed by insertion into a resident intramembranous binding site with few adaptative changes. Our binding data indicate that a balanced hydrophobicity and accurate positioning of the side chains, provided by the central guaianolide ring structure, defines a pharmacophore of Tg that governs both high affinity and access to the protein-binding site. Tg analogs substituted with long linkers at O-8 extend from the binding site between transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S. R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives with improved properties for SERCA targeting. Finally, we propose that the Tg binding pocket may be a regulatory site that, for example, is sensitive to cholesterol.

Studies of neuropeptide and peptide hormone signaling are coming of age in Drosophila due to rapid developments in molecular genetics approaches that overcome the difficulties caused by the small size of the fly. In addition we have genome-wide information on genes involved in peptide signaling, and growing pools of peptidomics data. A large number of different neuropeptides has been identified in a huge variety of neuron types in different parts of the Drosophila nervous system and cells in other locations. This review addresses questions related to peptidergic signaling in the Drosophila nervous system, especially how peptides regulate physiology and behavior during development and in the mature fly. We first summarize novel findings on neuropeptide precursor genes, processed bioactive peptides and their cognate receptors. Thereafter we provide an overview of the physiological and behavioral roles of peptide signaling in Drosophila. These roles include regulation of development, growth, feeding, metabolism, reproduction, homeostasis, and longevity, as well as neuromodulation in learning and memory, olfaction and locomotor control. The substrate of this signaling is the peptide products of about 42 precursor genes expressed in different combinations in a variety of neuronal circuits or that act as circulating hormones. Approximately 45 G-protein-coupled peptide receptors are known in Drosophila and for most of these the ligands have been identified. Functions of some peptides are better understood than others, and much work remains to reveal the spectrum of roles neuropeptides and peptide hormones play in the daily life of a fly., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.

The central complex is one of the most prominent neuropils in the insect brain. It has been implicated in the control of locomotor activity and is considered as a pre-motor center. Several neuropeptides are expressed in circuits of the central complex, and thus may be modulators of locomotor behavior. Here we have investigated the roles of two different neuropeptides, Drosophila tachykinin (DTK) and short neuropeptide F (sNPF), in aspects of locomotor behavior. In the Drosophila brain, DTK and sNPF are expressed in interneurons innervating the central complex. We have directed RNA interference (RNAi) towards DTK and sNPF specifically in different central complex neurons. We also expressed a temperature-sensitive dominant negative allele of the fly ortholog of dynamin called shibire(ts1), essential in membrane vesicle recycling and endocytosis, to disrupt synaptic transmission in central complex neurons. The spontaneous walking activity of the RNAi- or shibire(ts1)-expressing flies was quantified by video tracking. DTK-deficient flies displayed drastically increased center zone avoidance, suggesting that DTK is involved in the regulation of spatial orientation. In addition, DTK deficiency in other central complex neurons resulted in flies with an increased number of activity-rest bouts. Perturbations in the sNPF circuit indicated that this peptide is involved in the fine regulation of locomotor activity levels. Our findings suggest that the contribution of DTK and sNPF to locomotor behavior is circuit dependent and associated with particular neuronal substrates. Thus, peptidergic pathways in the central complex have specific roles in the fine tuning of locomotor activity of adult Drosophila.

Transfer and processing of olfactory information in the antennal lobe of Drosophila relies primarily on neurotransmitters such as acetylcholine and GABA, but novel studies also implicate a neuropeptide: the Drosophila tachykinin (DTK). DTK is expressed in local interneurons that innervate the glomeruli of the antennal lobe with varicose processes. Recently, DTK was shown to mediate presynaptic inhibition of olfactory sensory neurons by physiological and behavioral analysis.(1) That study drew our attention to the issue of alternative targets of DTK in the antennal lobe. Hence, in the present study, we interfered with DTK peptide and DTK receptor (DTKR) expression in local interneurons of the antennal lobe and studied the behavioral outcome of these manipulations. We show that DTKR is expressed not only in olfactory sensory neurons, but likely also in local interneurons. The behavioral consequences of interfering with postsynaptic peptide receptors are different from presynaptic peptide receptor interference. We discuss the possibility that the sum of pre- and postsynaptic interactions may be to modulate the dynamic range in odor sensitivity.

Improvements in the handling of membrane proteins for crystallization, combined with better synchrotron sources for X-ray diffraction analysis, are leading to clarification of the structural details of an ever increasing number of membrane transporters and receptors. Here we describe how this development has resulted in the elucidation at atomic resolution of a large number of structures of the sarcoplasmic Ca(2+)-ATPase (SERCA1a) present in skeletal muscle. The structures corresponding to the various intermediary states have been obtained after stabilization with structural analogues of ATP and of metal fluorides as mimicks of inorganic phosphate. From these results it is possible, in accordance with previous biochemical and molecular biology data, to give a detailed structural description of both ATP hydrolysis and Ca(2+) transport through the membrane, to serve as the starting point for a fuller understanding of the pump mechanism and, in future studies, on the regulatory role of this ubiquitous intracellular Ca(2+)-ATPase in cellular Ca(2+) metabolism in normal and pathological conditions.

Glucose ingestion during exercise attenuates the release of the myokine interleukin-6 (IL-6) from working skeletal muscle, which results in a diminished increase in plasma IL-6. Interleukin-6 receptor alpha (IL-6Ralpha) expression in skeletal muscle is induced by acute exercise, mediated in part by an increased IL-6 concentration in the bloodstream. We hypothesized that endurance training would increase the density of IL-6Ralpha in skeletal muscle and that glucose ingestion would attenuate the effect. Nine subjects performed 10 weeks of one-legged knee-extensor training. They trained one leg (Glc-leg) while ingesting a glucose solution (Glc) and ingested a placebo (Plc) while training the other leg (Plc-leg). Endurance training increased peak power by 14% and reduced the exercise-induced gene expression of IL-6 and IL-6Ralpha in skeletal muscle and IL-6 plasma concentration. The IL-6Ralpha density increased to a lesser extent in the Glc-leg, suggesting that glucose ingestion attenuates the effect of training on IL-6Ralpha by blunting the IL-6 response. We conclude that glucose ingestion during endurance training attenuates the increase in IL-6Ralpha density.

Crystal diffraction of three membrane proteins (cytochrome bc(1) complex, sarcoplasmic reticulum Ca(2+) ATPase, ADP-ATP carrier) and of one nucleoprotein complex (leucyl tRNA synthetase bound to tRNAleu, leuRS:tRNAleu) was tested at wavelengths near the X-ray K-absorption edge of phosphorus using a new set-up for soft X-ray diffraction at the beamline ID01 of the ESRF. The best result was obtained from crystals of Ca(2+) ATPase [adenosin-5'-(beta,gamma-methylene) triphosphate complex] which diffracted out to 7 A resolution. Data were recorded at a wavelength at which the real resonant scattering factor of phosphorus reaches the extreme value of -20 electron units. The positions of the four triphosphates of the monoclinic unit cell of the ATPase have been obtained from a difference Fourier synthesis based on a limited set of anomalous diffraction data.

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antannal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). We show here that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, we found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. We detect opposite behavioral phenotypes after reduction and over expression of DTKR in ORNs. Our findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe.

The first crystal structure of the neurotransmitter/sodium symporter homolog LeuT revealed an occluded binding pocket containing leucine and 2 Na(+); later structures showed tricyclic antidepressants (TCAs) in an extracellular vestibule approximately 11 A above the bound leucine and 2 Na(+). We recently found this region to be a second binding (S2) site and that binding of substrate to this site triggers Na(+)-coupled substrate symport. Here, we show a profound inhibitory effect of n-octyl-beta-d-glucopyranoside (OG), the detergent used for LeuT crystallization, on substrate binding to the S2 site. In parallel, we determined at 2.8 A the structure of LeuT-E290S, a mutant that, like LeuT-WT, binds 2 substrate molecules. This structure was similar to that of WT and clearly revealed an OG molecule in the S2 site. We also observed electron density at the S2 site in LeuT-WT crystals, and this also was accounted for by an OG molecule in that site. Computational analyses, based on the available crystal structures of LeuT, indicated the nature of structural arrangements in the extracellular region of LeuT that differentiate the actions of substrates from inhibitors bound in the S2 site. We conclude that the current LeuT crystal structures, all of which have been solved in OG, represent functionally blocked forms of the transporter, whereas a substrate bound in the S2 site will promote a different state that is essential for Na(+)-coupled symport.

P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.

Objective: IL-15 decreases lipid deposition in preadipocytes and decreases the mass of white adipose tissue in rats, indicating that IL-15 may take part in regulating this tissue. IL-15 is expressed in human skeletal muscle and skeletal muscle may be a source of plasma IL-15 and in this way regulate adipose tissue mass., Design: The relation between skeletal muscle IL-15 mRNA expression, plasma IL-15, and adipose tissue mass was studied in 199 humans divided into four groups on the basis of obesity and type 2 diabetes. Furthermore, using a DNA electrotransfer model, we assessed the effect of IL-15 overexpression in skeletal muscle of mice., Results: In humans, multiple regression analysis showed a negative association between plasma IL-15 and total fat mass (P<0.05), trunk fat mass (P<0.01), and percent fat mass (P<0.05), independent of type 2 diabetes. Negative associations were also found between muscle IL-15 mRNA and obesity parameters. IL-15 overexpression in skeletal muscle of mice reduced trunk fat mass but not sc fat mass., Conclusions: Our results indicate that IL-15 may be a regulator of trunk fat mass.

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF(4)(-) bound (3.5-A resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-A resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca(2+) was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca(2+)-translocating Ca(2)E1P-->E2P transition, which seems to be due to a stabilization of Ca(2)E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro(312) with alanine in P312A.

The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.

Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.

The invertebrate tachykinin-related peptides (TKRPs) constitute a conserved family, structurally related to the mammalian tachykinins, including members such as substance P and neurokinins A and B. Although their expression has been documented in the brains of insects and mammals, their neural functions remain largely unknown, particularly in behavior. Here, we have studied the role of TKRPs in Drosophila. We have analyzed the olfactory perception and the locomotor activity of individuals in which TKRPs are eliminated in the nervous system specifically, by using RNAi constructs to silence gene expression. The perception of specific odorants and concentrations is modified towards a loss of sensitivity, thus resulting in a significant change of the behavioral response towards indifference. In locomotion assays, the TKRP-deficient flies show hyperactivity. We conclude that these peptides are modulators of olfactory perception and locomotion activity in agreement with their abundant expression in the olfactory lobes and central complex. In these brain centers, TKRPs seem to enhance the regulatory inhibition of the neurons in which they are expressed.

Proctolin was the first insect neuropeptide to be sequenced and has been the subject of many physiological and pharmacological studies in insects and crustaceans. We have identified a Drosophila gene (CG7105, Proct) encoding a precursor protein containing the neuropeptide proctolin (RYLPT). In situ hybridization with a riboprobe to the Proct gene revealed a distribution of transcript in neurons of the larval central nervous system (CNS) matching that seen with antiserum to proctolin. An antiserum raised to a sequence in the precursor downstream of proctolin labeled the same neurons as those seen with the antiproctolin antisera. The predicted protein encoded by Proct has a single copy of the RYLPT sequence that directly follows the predicted signal peptidase cleavage point and precedes a consensus recognition site for a furinlike processing endoprotease. Ectopic expression of Proct in the CNS and midgut via the GAL4-UAS system, using an Actin5C-GAL4 driver, confirmed that the predicted preproproctolin can be processed to generate immunoreactive proctolin peptide. Pupae over-expressing Proct displayed a 14% increase in heart rate, providing evidence in support of a cardioacceleratory endocrine function for proctolin in Drosophila. The distribution of proctolin suggests roles as a neuromodulator in motoneurons and interneurons, and as a neurohormone that could be released from brain neurosecretory cells with terminations in the ring gland., (Copyright 2003 Wiley Periodicals, Inc. J Neurobiol 58: 379-391, 2004)

The gene Dtk, encoding the prohormone of tachykinin-related peptides (TRPs), has been identified from Drosophila. This gene encodes five putative tachykinin-related peptides (DTK-1 to 5) that share the C-terminal sequence FXGXRamide (where X represents variable residues) as well as an extended peptide (DTK-6) with the C-terminus FVAVRamide). By mass spectrometry (MALDI-TOF-MS), we identified ion signals with masses identical to those of DTK-1 to 5 in specific brain regions. We have analyzed the distribution of the Dtk transcript and peptides, by in situ hybridization and immunocytochemistry during postembryonic development of the central nervous system (CNS) of Drosophila. Antiserum against a cockroach TRP that cross-reacts with the DTKs was used for immunocytochemistry. Expression of transcript and peptides was detected from first to third instar larvae, through metamorphosis to adult flies. Throughout postembryonic development, we were able to follow the strong expression of TRPs in a pair of large descending neurons with cell bodies in the brain. The number of TRP-expressing neuronal cell bodies in the brain and ventral nerve cord increases during larval development. In the early pupa (stage P8), the number of TRP-expressing cell bodies is lower than in the third instar larvae. The number drastically increases during later pupal development, and in the adult fly about 200 TRP-expressing neurons can be seen in the CNS. The continuous expression of TRPs in neurons throughout postembryonic development suggests specific functional roles in both larval and imaginal flies and possibly also in some neurons during pupal development., (Copyright 2003 Wiley-Liss, Inc.)

Seven isoforms of tachykinin-related peptides (TRPs) have been isolated from the brain of the cockroach Leucophaea maderae. These peptides (LemTRP-1, 2, and 5-9) share the C-terminal sequence GFX(1)GX(2)Ramide (where X(1) and X(2) are variable residues). In order to determine the neuronal distribution of several of these LemTRP isoforms, we raised antisera to their variable N-termini. Antisera to LemTRP-1, 2, 3, 7, and 8 were utilized for immunocytochemistry on cryostat sections of the L. maderae brain. As expected, the gut peptide LemTRP-3 was not detected in the brain, and the antisera to LemTRP-1, 2, and 7 labeled the same sets of neurons in different regions of the brain. These neurons could also be labeled with antisera raised to the more conserved C-termini of LemTRP-1 and the locust TRP LomTK-I. The antiserum to LemTRP-8 predominantly labeled a set of neurons distinct from that seen with any other N- or C-terminus-directed antisera, suggesting that it recognizes epitope(s) other than known insect TRPs. Our findings indicate that at least three of the LemTRPs are always co-localized in neurons of the L. maderae brain. We have also been able to show that LemTRP-2, which is an N-terminally extended form (17-mere) of LemTRP-1 with a dibasic putative cleavage site, is transported throughout the processes of the neurons in the same manner as LemTRP-1 and 7. Thus, LemTRP-2 may be released with the other shorter LemTRPs. This is the first investigation of LemTRP distribution in the cockroach central nervous system utilizing antisera to native peptides.

Tachykinin-related peptides (TRPs) in the locust Locusta migratoria and the cockroach Leucophaea maderae have stimulatory effects on some muscles that are not innervated by TRP-containing neurons. Thus, these tissues may be affected by circulating TRPs. Here, we have investigated whether the midgut is the source of circulating TRPs. TRP-immunoreactive material in the locust midgut is found only in the endocrine cells of the gut epithelium. In both species of insect, the endocrine cells contain several isoforms of TRPs, as determined by immunocytochemistry and a combination of chromatography (HPLC) and enzyme immunoassay (ELISA). The release of TRPs was investigated by ELISA using isolated midguts of the locust and cockroach. Elevated levels of K(+) in the bathing saline induced the release of TRP from the midgut of both species. To examine the release of TRPs into the circulation in vivo, we measured haemolymph levels of TRPs in fed and starved locusts. The concentration of TRP-immunoreactive material in fed locusts was estimated to be 0.15 nmol l(-1), and this increased approximately fourfold in insects starved for 24 h. In accordance with this observation, the content of TRP-immunoreactive material in the midgut was lower in starved locusts than in fed locusts. Although part of the increased blood concentration of TRPs may be due to reduced blood volume, our data suggest that TRPs are released as hormones from the midgut of the locust and cockroach and that this release may be linked to nutritional status.

The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction). Chemically, the allatostatins can be subdivided into three different peptide groups: the large group of A-type (cockroach-type) allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins, which have the C-terminal sequence W(X(6))Wamide in common; and a single allatostatin that we now call C-type allatostatin that was first discovered in the moth Manduca sexta, and which has a nonamidated C terminus, and a structure unrelated to the A- and B-type allatostatins. We have previously cloned the preprohormones for the A- and B-type allatostatins from Drosophila melanogaster. Here we report on the cloning of a Drosophila C-type allatostatin preprohormone (DAP-C). DAP-C is 121 amino acid residues long and contains one copy of a peptide sequence that in its processed form has the sequence Y in position 4) from the Manduca sexta C-type allatostatin. The DAP-C gene has three introns and four exons and is located at position 32D2-3 on the left arm of the second chromosome. Northern blots show that the gene is strongly expressed in larvae and adult flies, but less in pupae and embryos. In situ hybridizations of larvae show that the gene is expressed in various neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera (flies)., (Copyright 2001 Academic Press.)

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2),

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Five isoforms of tachykinin-related peptides (TRPs), designated LemTRP-1-5, have been identified in the midgut of the cockroach Leucophaea maderae. These peptides have a conserved C-terminus hexapeptide (GFX1GX2Ramide; X1 and X2 are variable residues) and variable N-termini. Here, we address the question of whether these five isoforms are all colocalized in the two types of cells in the cockroach midgut, the endocrine cells and the neuronal processes. We also investigate whether the N-terminally extended isoforms LemTRP-2 and -3, which contain putative endoproteolytic cleavage sites, are expressed in intact form or are cleaved in the midgut cells. To this end, we used two approaches. (1) Extracts from portions of the midgut containing each of the cell types were subjected to reverse-phase high performance liquid chromatography (HPLC) and the fractions monitored in a radioimmunoassay (RIA) with an antiserum to the conserved C-terminus of insect TRPs. (2) Antisera were raised to the variable N-termini of the extended LemTRP-2 and -3 and used for immunocytochemistry. The HPLC-RIA and immunocytochemical findings indicate that LemTRP-1 and 4-5 are present in the neuronal processes and in endocrine cells of the midgut proper and of the gastric cecae. The two extended forms LemTRP-2 and -3 display a differential distribution: LemTRP-2 was found in endocrine cells of midgut and gastric cecae, but not in neuronal processes, whereas LemTRP-3 was seen in neuronal processes and endocrine cells of the midgut proper, but not in the gastric cecae. LemTRP-3 and -4 have not been identified in the brain, suggesting further cell- and tissue-specific expression of LemTRPs. The mechanisms behind the cell-specific expression of the LemTRPs are not yet understood, but the demonstration of differential distribution of the peptide isoforms provide a first indication that the isoforms may have different actions.

The nine Leucophaea Tachykinin-Related Peptides (LemTRP 1-9) isolated from the midgut and brain of the cockroach, Leucophaea maderae, all induced increases in spontaneous contractions of the L. maderae hindgut. Synthetic LemTRP 1 and 3-9, were equally potent in inducing contractions of the hindgut. More than seven of the nine C-terminal residues of the closely related locust peptide locustatachykinin I (LomTK I) are required for full activity of the peptide on the L. maderae hindgut. Proctolin, a well characterized myostimulatory neuropeptide, was shown to be more potent than LemTRPs. LemTRP 1 and proctolin did not have synergistic actions in potentiating the amplitude and tonus of contractions of the L. maderae hindgut. Several differences could be seen in actions of LemTRP 1 and proctolin. In contrast to proctolin, LemTRP 1 could not override the inhibitory action of 10(-9) M of the myoinhibitory peptide leucomyosuppressin. Spantide I, an antagonist of the mammalian tachykinin receptors, at a concentration of 5 microM, blocked the response to LemTRP 1, but not to proctolin. The competitive proctolin receptor antagonist [alpha-methyl-L-tyrosine2]-proctolin blocked the action of both proctolin and LemTRP 1 when applied at 1 microM, whereas cycloproctolin had no antagonist action on either peptide. Verapamil, a blocker of voltage gated Ca2+-channels, and the less specific Ca2+-channel blocker Mn2+, abolished the action of LemTRP 1, but not of proctolin. The results obtained indicate that LemTRPs act on receptors distinct from those of proctolin. Double label immunocytochemistry revealed that all LomTK-like immunoreactive fibers impinge on the proctolinergic fibers in the hindgut. This finding and the inhibitory actions of Ca2+-channel blockers on TRP responses and of the proctolin receptor antagonist on both peptides, may suggest that the LemTRP receptors are not on the hindgut muscle fibers but on the terminals of the proctolinergic neurons. Thus, LemTRPs may induce release of proctolin on the hindgut. An alternative is that LemTRPs act by mechanisms clearly distinct from those of proctolin.

Two neurohemal organs of the cockroach Leucophaea maderae, the corpora cardiaca and the lateral heart nerve are known to contain leucokinin immunoreactive material. We examined the corpora cardiaca and the lateral heart nerve to establish whether these neurohemal organs store all 8 known leucokinin isoforms or if the leucokinins have a differential distribution. Extracts of corpora cardiaca and abdominal hearts with attached lateral heart nerve were separated on reversed phase high performance liquid chromatography (rpHPLC), then tested for leucokinin immunoreactivity by a radioimmunoassay (RIA) able to detect all 8 leucokinin isoforms. Extracts from brain and optic lobes were also separated and assayed in the RIA. Synthetic leucokinin 1-8 were subjected to rpHPLC and their different retention times established by RIA for reference. Leucokinin immunoreactive material originating from the corpora cardiaca and lateral heart nerves eluted in fractions corresponding to those of the synthetic leucokinin 1-8. In this study we have thus demonstrated that probably all 8 leucokinin isoforms are stored in the corpora cardiaca and the lateral heart nerve. These observations suggest that all 8 leucokinins are likely to be released as neurohormones into the circulation.