Expression of recombinant eyestalk crustacean hyperglycemic hormone from the tropical land crab, <Emphasis Type="Italic">Gecarcinus lateralis</Emphasis>, that inhibits Y-organ ecdysteroidogenesis in vitro

Crustacean hyperglycemic hormone (CHH) is a pleiotropic neuropeptide that regulates carbohydrate and lipid metabolism, molting, reproduction, and osmoregulation in decapod crustaceans. CHH elevates glucose levels in the hemolymph by stimulating glycogenolysis in target tissues. It also inhibits ecdysteroidogenesis in the molting gland, or Y-organ (YO), possibly as a response to environmental stress. CHH acts via binding to a membrane receptor guanylyl cyclase, which is expressed in most tissues, including the YO. Large amounts of biologically active neuropeptide are required to investigate the mechanism of CHH signaling in the YO. Consequently, the eyestalk ganglia CHH (EG-CHH) isoform was cloned into a yeast (Pichia pastoris) expression vector to express recombinant mature peptide (rEG-CHH) with or without a C-terminal c-Myc/polyhistidine tag. Yeast cultures with untagged or tagged rEG-CHH inhibited ecdysteroidogenesis in YOs from European green crab (Carcinus maenas) 36% (P < 0.002) and 51% (P < 0.006), respectively. Purified tagged EG-CHH inhibited YO ecdysteroidogenesis 32% (P < 0.002), but lacked hyperglycemic activity in vivo. This is the first report of recombinant EG-CHH inhibiting YO ecdysteroidogenesis. The data suggest that the tagged recombinant peptide can be used to elucidate the CHH signaling pathway in the crustacean molting gland.     

Demonstration of the cellular expression of genes encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in the eyestalk of the shore crab Carcinus maenas

Based on the amino acid sequence of the molt-inhibiting hormone of Carcinus maenas, two degenerated oligonucleotide primers were synthesized and used in the polymerase chain reaction. By use of complementary DNA of a library constructed from medulla terminalis-X-organ RNA of C. maenas as template, the specific complementary DNA between the primers was amplified, cloned and sequenced. This strategy revealed a DNA sequence for which the deduced amino acid sequence is identical to the recently published C. maenas molt-inhibiting hormone sequence as determined by Edman degradation. Visualization of messenger RNAs encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in different perikarya of the X-organ was obtained using digoxigenin-labelled complementary RNA probes. Combination of immunocytochemical staining using polyclonal antisera against the native C. maenas neuropeptides and in situ hybridization performed on alternating sections confirmed the specificity of the reaction. The results show that there is no co-localization of molt-inhibiting hormone and crustacean hyperglycemic hormone at the messenger RNA and the protein level.     

Posttranslational isomerization of a neuropeptide in crustacean neurosecretory cells studied by ultrastructural immunocytochemistry

Isomerization of the third amino acid residue (a phenylalanine) of crustacean hyperglycemic hormone (CHH) has been previously reported to occur as a late step of hormone precursor maturation in a few neurosecretory cells in the X-organ-sinus gland complex of the crayfish Orconectes limosus. In the present report, using conformation-specific antisera combined with immunogold labeling, we have studied, at the ultrastructural level, the distribution of L- and D-CHH immunoreactivity in CHH-secreting cells of the crayfish Astacus leptodactylus. Two CHH-secreting cell populations were observed, the first one (L-cells), the most numerous, exhibited only labeling for L-CHH. In the second one (D-cells), four secretory granule populations were distinguished according to their labeling: unlabeled, either L- or D- exclusively or both L- and D-granules. Labeling quantification by image analysis in D-cells showed a marked increase in D-labeling from the cell body to the axon terminal. However some L- and mixed granules remain in axon terminals. Our results demonstrate that Phe3 isomerization of CHH occurs within the secretory granules of specialized neurosecretory cells and progresses as the granules migrate along the axonal tract. The observation that not all the CHH synthesized is isomerized, and the great variability in the proportion of L- and D-immunoreactivity in granules in every cell region may suggest an heterogeneous distribution of the putative enzyme involved in Phe3 isomerization, a peptide isomerase, within the secretory pathway.     

Identification of neuropeptides from the decapod crustacean sinus glands using nanoscale liquid chromatography tandem mass spectrometry

Neurosecretory systems are known to synthesize and secrete a diverse class of peptide hormones which regulate many physiological processes. The crustacean sinus gland (SG) is a well-defined neuroendocrine site that produces numerous hemolymph-borne agents including the most complex class of endocrine signaling molecules--neuropeptides. As an ongoing effort to define the peptidome of the crustacean SG, we determine the neuropeptide complements of the SG of the Jonah crab, Cancer borealis, and the Maine lobster, Homarus americanus, using nanoflow liquid chromatography electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS. Numerous neuropeptides were identified, including orcokinins, orcomyotropin, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptides (CPRPs), red pigment concentrating hormone (RPCH), beta-pigment dispersing hormone (beta-PDH), proctolin and HL/IGSL/IYRamide. Among them, two novel orcokinins were de novo sequenced from the SG of H. americanus. Three CPRPs including a novel isoform were sequenced in H. americanus. Four new CPRPs were sequenced from the SG of C. borealis. Our results show that structural polymorphisms in CPRPs (and thus the CHH precursors) are common in Dendrobranchiata as well as in Pleocyemata. The evolutionary relationship between the CPRPs is also discussed.     

Molecular biology of neurohormone precursors in the eyestalk of Crustacea

Our knowledge concerning the primary structures of crustacean neuropeptides has been broadened considerably during the last few years and has greatly contributed to the successful application of molecular biological techniques to crustacean neuroendocrine research. In this review, we compare and discuss the preprohormones of the Red Pigment Concentrating Hormone (RPCH), the Pigment-Dispersing Hormone (PDH) and the different members of the Crustacean Hyperglycemic Hormone, Molt-Inhibiting and Gonad-Inhibiting Hormone family (CHH/MIH/ GIH peptide family), recently elucidated by cloning and sequencing of the respective cDNAs. Expression studies, using in situ hybridization, Northern blots and RNase protection assays, have demonstrated that the mRNAs encoding some of the aforementioned preprohormones (for example, preproPDH and preproCHH) are not only expressed in the eyestalk but also in other parts of the central nervous system. The combination of molecular biological techniques with (bio)chemical and immunochemical methods provides elegant tools to study neuropeptides at the level of mRNA and peptide in individual animals during different physiological conditions. The fundamental knowledge obtained by such a combined approach will give detailed insight into how neuropeptides are involved in the adaptation of Crustacea to a broad spectrum of natural and aquacultural conditions.     

Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site

The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.     

Non-target effects of the insecticide methoprene on molting in the estuarine crustacean Neomysis integer (Crustacea: Mysidacea)

Ecdysteroids, the molting hormones in crustaceans and other arthropods, play a crucial role in the control of growth, reproduction and embryogenesis of these organisms. Insecticides are often designed to target specific endocrine-regulated functions such as molting and larval development such as methoprene, a juvenile hormone analogue.The aim of this study was to examine the effects of methoprene on molting in a non-target species, the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Mysids have been proposed as standard test organisms for evaluating the endocrine disruptive effect of chemicals. Juveniles (< 24 h) were exposed for 3 weeks to the nominal concentrations 0.01, 1 and 100 μg methoprene/l. Daily, present molts were checked and stored in 4% formaldehyde for subsequent growth measurements. Methoprene significantly delayed molting at 100 μg/l by decreasing the growth rate and increasing the intermolt period. This resulted in a decreased wet weight of the organism. The anti-ecdysteroidal properties of methoprene on mysid molting were also evaluated by determining the ability of exogenously administered 20-hydroxyecdysone, the active ecdysteroid in crustaceans, to protect against the observed methoprene effects. Co-exposure to 20-hydroxyecdysone did not mitigate methoprene effects on mysid molting. This study demonstrates the need for incorporating invertebrate-specific hormone-regulated endpoints in regulatory screening and testing programs for the detection of endocrine disruption caused by man-made chemicals.     

Neuroendocrine stimulation of uterine gland protein synthesis in the tsetse fly, Glossina morsitans

ABSTRACT. The uterine gland of the tsetse fly Glossina morsitans morsitans Westw. synthesizes a secretion which nourishes the developing larva in the uterus. Aqueous extracts of the brain have been shown to stimulate the synthesis of the protein and amino acid components of this secretion from L- [U-¹⁴C]leucine by uterine gland tubules in vivo and in vitro. A linear dose response relationship was demonstrated in vitro with extract concentrations ranging from 1 × 10^-4 to 1 × 10^-2 brains μl^-1. The maximum response, a > 300% increase in the rate of protein and amino acid synthesis, was achieved with as little as 1 × 10^-2 brains μl^-1 The concentration of active factor(s) in the brain declined during a single interlarval period coincident with the period of release of secretion associated with larval growth. The stimulatory activity in brain extracts was destroyed by proteolytic enzymes indicating that it is probably a protein or peptide. Results suggest that the active factor(s) is a hormone responsible for the stimulation of uterine gland protein synthesis essential for larval nutrition.     

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna, erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC₅₀s) of 2.8 · 10⁻¹¹ mol · l⁻¹, 7.0 · 10⁻⁷ mol · l⁻¹ and 3.3 · 10⁻⁷ mol · l⁻¹, respectively. KT5720 (10⁻⁷ mol · l⁻¹ and 10⁻⁶ mol · l⁻¹) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10⁻⁴ mol · l⁻¹ of adenosine and adenosine triphosphate (ATP), and up to 10⁻³ mol · l⁻¹ of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10⁻⁷ mol · l⁻¹) and forskolin (10⁻⁶ mol · l⁻¹), but were not affected by PCH (10⁻⁷ or 10⁻¹⁰ mol · l⁻¹). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated. Accepted: 11 August 1998     

Secretory stages of individual CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus,determined by means of immunocytochemistry

Summary Immunocytochemical staining demonstrates striking differences in staining intensity among individual crustacean hyperglycemic hormone (CHH)-producing cells in the eyestalk of the crayfish Astacus leptodactylus. Based on these differences we arbitrarily subdivided the CHH-cells into three categories representing increasing immunoreactivity respectively: + cells, + + cells, and + + + cells. Electron microscopic investigations reveal that these differences in immunostaining are correlated with differences in the numerical density of the neurosecretory granules in the cytoplasm and that these may reflect differences in activity among the CHH-cells. Morphometric analyses at the light- and electron-microscopic levels indicate that the three distinguished categories of immunopositive cells represent different stages in the CHH-synthesizing process of the cells. The results of the present study demonstrate the application of the PAP-technique at the light-microscopic level as a method to obtain information pertaining to the dynamics of secretory activity of the CHH-cells.     

Modulation of inwardly rectifying Na+-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech,Haementeria

Summary The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about –70 mV were associated with the activation of a slow inward current (I _h) which showed no inactivation with time. The time course of activation of I _hwas described by a single-exponential function and was strongly voltage dependent. The activation curve of_hranged from –72 to –118 mV, with half-activation occurring at –100 mV. Ion-substitution experiments indicated that I _his carried by both Na⁺ and K⁺ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of I _hand its rale of activation. It also produced a positive shift of the activation curve of the conductance underlying I _h G_hwithout altering the slope factor, thus indicating that the voltage dependence of I _hwas modulated by 5-HT. Cs⁺ blocked both I _hand the 5-HT-polentiated current in a voltage-independent manner, whereas Ba²⁺ had little effect. It is concluded that 5-HT increases I _hby modulating the inwardly rectifying Na⁺-K⁺ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since I _hwas increased by 8-bromocyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine. In contrast, I _hwas reduced by 8-bromo-cyclic GMPand by zaprinast (an inhibitor of cyclic GMP-scnsitive phosphodieslerase). Cyclic GMP itself also reduced I _h, and the effect was specific to the 3,5 form; 2,3-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMPThis work was supported by a grant from the Science and Engineering Research Council (no. GR/F/17087). We are grateful to the SmithKline (1982) Foundation for provision of a pulse generator     

Conserved role of PROTON GRADIENT REGULATION 5 in the regulation of PSI cyclic electron transport

There are at least two photosynthetic cyclic electron transport (CET) pathways in most C₃ plants: the NAD(P)H dehydrogenase (NDH)-dependent pathway and a pathway dependent upon putative ferredoxin:plastoquinone oxidoreductase (FQR) activity. While the NDH complex has been identified, and shown to play a role in photosynthesis, especially under stress conditions, less is known about the machinery of FQR-dependent CET. Recent studies indicate that FQR-dependent CET is dependent upon PGR5, a small protein of unknown function. In a previous study we found that overexpression of PGR5 causes alterations in growth and development associated with decreased chloroplast development and a transient increase in nonphotochemical quenching (NPQ) after the shift from dark to light. In the current study we examine the spatiotemporal expression pattern of PGR5, and the effects of overexpression of PGR5 in Arabidopsis under a host of light and stress conditions. To investigate the conserved function of PGR5, we cloned PGR5 from a species which apparently lacks NDH, loblolly pine, and overexpressed it in Arabidopsis. Although greening of cotyledons was severely delayed in overexpressing lines under low light, mature plants survived exposure to high light and drought stress better than wild-type. In addition, PSI was more resistant to high light in the PGR5 overexpressors than in wild-type plants, while PSII was more sensitive to this stress. These complex responses corresponded to alterations in linear and cyclic electron transfer, suggesting that over-accumulation of PGR5 induces pleiotropic effects, probably via elevated CET. We conclude that PGR5 has a developmentally-regulated, conserved role in mediating CET.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

Physiological investigation on the role of the innervation in the regulation of the prothoracic gland in Periplaneta americana

Under in vitro conditions the prothoracic gland nerve of the last larval instar of Periplaneta americana shows the same efferent nervous activity as under in situ conditions–ie, low activity at the 9th day and high activity at the 20th day of the molting interval. Isolation of the prothoracic ganglion from the subesophageal ganglion provokes an increase in this nerve activity, suggesting an inhibitory effect of the subesophageal ganglion on prothoracic gland nerve activity in vivo. Only in 20-day-old larvae does electrical stimulation of isolated prothoracic glands in vitro via the gland nerve result in a slightly increased release of ecdysteroids from the gland. This effect could not be influenced by different lengths of stimulation periods. Denervation of the prothoracic gland by transection of the gland nerve on the 13th day of the molting interval results in a complete abolition of the first peak of ecdysteroid production in the gland but has no influence on the occurrence and the amount of the main ecdysteroid peak just before the molt. The results suggest the participation of nervous activity in special periods of prothoracic gland regulation in the cockroach.