IDENTIFICATION OF THE FGL‐AMIDE ALLATOSTATIN GENE OF THE PRIMITIVE TERMITE Mastotermes darwiniensis AND THE WOODROACH Cryptocercus darwini

Allatostatins with the C‐terminal ending Tyr/Phe‐Xaa‐Phe‐Gly‐Leu/Ile‐amide (FGLa/ASTs) are widespread neuropeptides with multiple functions. The gene encoding the FGLa/AST polypeptide precursor was first isolated from cockroaches and since then could be identified in many insects and crustaceans. With its strictly conserved regions in combination with variable regions the gene seems to be a good candidate for phylogenetic analyses between closely and distantly related species. Here, the structure of the FGLa/AST gene of the most primitive termite, the giant northern termite Mastotermes darwiniensis Froggatt, was identified. The FGLa/AST gene of the woodroach Cryptocercus darwini was also determined. Precursor sequences of both species possess the general organization of dictyopteran FGLa/AST precursors containing 14 putative FGLa/AST peptides. In M. darwiniensis, only 11 out of the 14 FGLa/AST‐like peptides possess the C‐terminal conserved region Y/FXFGL/I/V/M and four of the putative peptide structures are not followed by a Gly residue that would lead to nonamidated peptides. Phylogenetic analyses show the high degree of similarity of dictyopteran FGLa/AST sequences. The position of termites, nested within the Blattaria, confirms that termites have evolved from primitive cockroaches.     

Influence of cholecystokinin on central monoaminergic pathways

Dopamine (DA) and cholecystokinin octapeptide carboxy-terminal (CCK-8) have been found to coexist in some mesolimbic neurons. The present investigation was undertaken in order to study the biochemical and behavioral interactions between CCK-8 and some central monoaminergic pathways. The action of the sulfated form of CCK-8 (10 micrograms/10 microliter intracerebroventricularly) on DA turnover in nucleus accumbens, olfactory tubercles and corpus striatum of the rat was determined after DA synthesis inhibition with alpha-methyl-p-tyrosine (250 mg/kg i.p.). Also, CCK-8 action (1-30 micrograms intracisternally) on DA synthesis was assessed by measuring accumulation of dihydroxyphenylalanine (DOPA) after DOPA-decarboxylase inhibition with NSD-1015 (m-hydroxybenzylhydrazine, 100 mg/kg i.p.). The contents of DA and its main metabolites, dihydroxyphenylacetic acid and homovanillic acid, together with serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were measured in different brain areas after direct injection of CCK-8 into the ventral tegmental area (A10) or nucleus accumbens. Further, the effect of CCK-8 on amphetamine-induced locomotion and apomorphine-induced stereotypies was studied along with changes in spontaneous locomotion and rearing after CCK-8 injection into the ventral tegmental area and nucleus accumbens. No consistent statistically significant effects of CCK-8 on biochemical or behavioral assessments on measures of DA function were observed. However, injection of high doses of CCK-8 into the ventral tegmental area significantly decreased levels of 5-HIAA in the nucleus accumbens, olfactory tubercles and striatum.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

High-performance liquid chromatographic mapping and structural characterization of neurophysin isoforms

Both ion-exchange and reverse-phase HPLC protocols for micromapping of neurophysins have been examined and the structural relationships among the major isoforms identified in the maps have been characterized. Reverse-phase HPLC was found to be especially useful for obtaining fingerprints of the isoforms within each of the two major families of neurophysins, I (oxytocin-related) and II (vasopressin-related), for both bovine and human neurophysins from posterior pituitary sources. From fractionation of the bovine proteins on octylsilyl columns, at least four neurophysins I were identified, one of which corresponds to the intact sequence of 93 residues and three of which vary from the parent by various degrees of carboxyl-terminal truncation. For bovine neurophysin II, two isoforms were identified in the reverse-phase HPLC maps, both of 95 residues, which vary from one another by the residue, either Ile or Val, at position 89. Isoforms were also detected for human neurophysins, including a carboxyl-terminal truncated form of human neurophysin II. All of the major neurophysin isoforms and several of the minor forms were shown to be functionally active as expressed by their binding to peptide ligand affinity matrices. Reverse-phase HPLC mapping on the octylsilyl matrix allowed neurophysin fingerprinting of crude tissue extracts by providing a narrow "window" within which the neurophysins elute but many other polypeptides expected to be present are excluded. The reverse phase HPLC method provides a useful way to obtain isolated neurophysin isoforms for physicochemical characterizations now usually carried out with mixtures of isoforms obtained by ion-exchange chromatography. The method also has characteristics amenable both for high-sensitivity fingerprinting of neurophysin isoforms, from different species and anatomical sources, and as a prelude to microstructural and -functional characterization of the isoforms so isolated.     

Prolactin receptor in regulation of neuronal excitability and channels

Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca²⁺ influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca²⁺-dependent K⁺ channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL.     

Neuropeptides of the cotton fleahopper, Pseudatomoscelis seriatus (Reuter)

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter), is an economically important pest of cotton, and increasing concerns over resistance, detrimental effects on beneficial insects and safety issues associated with traditional insecticide applications have led to an interest in research on novel, alternative strategies for control. One such approach requires a more basic understanding of the neurohormonal system that regulates important physiological properties of the fleahopper; e.g. the expression of specific messenger molecules such as neuropeptides. Therefore we performed a peptidomic study of neural tissues from the fleahopper which led to the first identification of the sequences of native peptide hormones. These peptide hormones include the following neuropeptides: corazonin, short neuropeptide F (sNPF), myosuppressin, CAPA-pyrokinin and CAPA-PVK peptides. The CAPA-pyrokinin, sNPF, and CAPA-PVK peptides represent novel sequences. A comparison of fleahopper neuropeptides with those of related heteropteran species indicates that they are quite different. The sNPF of P. seriatus shows, among others, a novel substitution of Leu with Phe within the C-terminal region; a modification that sets it apart from the known sNPFs of not only other Heteroptera but of other arthropod species as well. The identity of the neuropeptides native to the fleahopper can aid in the potential development of biostable, bioavailable mimetic agonists and antagonists capable of disrupting the physiological functions that these neuropeptides regulate.     

Beyond the connectome: how neuromodulators shape neural circuits

Powerful ultrastructural tools are providing new insights into neuronal circuits, revealing a wealth of anatomically-defined synaptic connections. These wiring diagrams are incomplete, however, because functional connectivity is actively shaped by neuromodulators that modify neuronal dynamics, excitability, and synaptic function. Studies of defined neural circuits in crustaceans, C. elegans, Drosophila, and the vertebrate retina have revealed the ability of modulators and sensory context to reconfigure information processing by changing the composition and activity of functional circuits. Each ultrastructural connectivity map encodes multiple circuits, some of which are active and some of which are latent at any given time.     

Intracellular Localization of the HCS2 Gene Products in Identified Snail Neurons In Vivo and In Vitro

SUMMARY 1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca²⁺ concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy.2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system.3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons.4. The immunogold particles were practically absent in the bodies of cultured neurons.5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.     

Pharynx regeneration in planarians

The obtained and published data on pharynx regeneration in planarians have been reviewed. Planarians can regenerate from a small body fragment and restore all missing organs including the pharynx. The pharynx is a relatively autonomous organ with a differentiated structure and specialized function. Pharynx regeneration has specific features, and its studies are of considerable theoretical interest. Pharynx regeneration can also be a convenient model to study the molecular mechanisms of regeneration that remain undisclosed. In addition, this model can be used to test biologically active compounds in order to elucidate their effect on morphogenesis. This subject of investigation benefits by a simpler and more adequate analysis as well as a possibility to use large numbers of animals and small quantities of analyzed substances.     

Receptor assay guided structure‐activity studies of helicokinin insect neuropeptides and peptidomimetic analogues

Neuropeptides control numerous physiological processes in insects. The regulation of water balance is a crucial aspect of homeostasis in terrestrial insects and has been shown to be under endocrine control, primarily by corticotrophin releasing factor (CRF)‐related peptides and kinins. For helicokinin I, a diuretic neuropeptide from the economically important insect pest Heliothis virescens, detailed structure‐activity relationships have been established based on truncated structures, diverse amino acid scans and peptidomimetic analogues. The activities of selected compounds on functional expressed helicokinin receptors are compared with the results of a Malphigian tubule assay. Implications for further peptidomimetic variations are provided. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.