Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone.

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.

Identification and developmental expression of mRNAs encoding crustacean cardioactive peptide (CCAP) in decapod crustaceans.

Full-length cDNAs encoding crustacean cardioactive peptide (CCAP) were isolated from several decapod (brachyuran and astacuran) crustaceans: the blue crab Callinectes sapidus, green shore crab Carcinus maenas, European lobster Homarus gamarus and calico crayfish Orconectes immunis. The cDNAs encode open reading frames of 143 (brachyurans) and 139-140 (astacurans) amino acids. Apart from the predicted signal peptides (30-32 amino acids), the conceptually translated precursor codes for a single copy of CCAP and four other peptides that are extremely similar in terms of amino acid sequence within these species, but which clearly show divergence into brachyuran and astacuran groups. Expression patterns of CCAP mRNA and peptide were determined during embryonic development in Carcinus using quantitative RT-PCR and immunohistochemistry with whole-mount confocal microscopy, and showed that significant mRNA expression (at 50% embryonic development) preceded detectable levels of CCAP in the developing central nervous system (CNS; at 70% development). Subsequent CCAP gene expression dramatically increased during the late stages of embryogenesis (80-100%), coincident with developing immunopositive structures. In adult crabs, CCAP gene expression was detected exclusively in the eyestalk, brain and in particular the thoracic ganglia, in accord with the predominance of CCAP-containing cells in this tissue. Measurement of expression patterns of CCAP mRNA in Carcinus and Callinectes thoracic ganglia throughout the moult cycle revealed only modest changes, indicating that previously observed increases in CCAP peptide levels during premoult were not transcriptionally coupled. Severe hypoxic conditions resulted in rapid downregulation of CCAP transcription in the eyestalk, but not the thoracic ganglia in Callinectes, and thermal challenge did not change CCAP mRNA levels. These results offer the first tantalising glimpses of involvement of CCAP in environmental adaptation to extreme, yet biologically relevant stressors, and perhaps suggest that the CCAP-containing neurones in the eyestalk might be involved in adaptation to environmental stressors.

Binding sites of crustacean hyperglycemic hormone and its second messengers on gills and hindgut of the green shore crab, Carcinus maenas: a possible osmoregulatory role.

To determine the possible involvement of crustacean hyperglycemic hormone (CHH) in osmoregulation in crustaceans, ligand binding and second messenger assays were performed on gills and hindgut preparations of the green shore crab Carcinus maenas, whilst midgut gland, previously known as one of the target tissues of CHH served as a control tissue. Classical receptor binding analyses using [(125)I]CHH by saturation and displacement experiments from membrane preparations from gills, hindgut, and midgut glands demonstrated that CHH binding characteristics involved one site, highly specific, saturable, and displaceable kinetics: (gills: K(D) 5.87 +/- 2.05 x 10(-10) and B(MAX) 6.50 +/- 1.15 x 10(-10), hindgut: K(D) 3.54 +/- 1.49 x 10(-10) and B(MAX) 2.31 +/- 0.44 x 10(-10), and midgut gland: K(D) 7.28 +/- 0.9 x 10(-10) and B(MAX) 3.28 +/- 0.25 x 10(-10)) all expressed as M/mg protein. No differences, in terms of displacement were observed between the two CHH isoforms (N-terminally blocked pGlu and unblocked Gln) variants. CHH binding sites appeared to be coupled to a second messenger system involving cGMP in all the tissues examined. Exposure of crabs to dilute seawater increased levels of cGMP, glucose in gills and circulating CHH levels. Other crustacean neuropeptides including crustacean cardioactive peptide, molt inhibiting hormone, L-enkephalin, FMRF-amide, proctolin, and crustacean hyperglycemic hormone precursor-related peptide were tested with regard to possible osmoregulatory roles with reference to changes in second messenger (cAMP and cGMP) concentrations in gill, hindgut, and midgut tissues in vitro, following application at 2 x 10(-8) M but all were found to be inactive. Thus, it seems likely that CHH is a pertinent neurohormone involved in osmoregulation, thus expanding its many functions as a pleiotropic hormone in crustaceans.

Dynamics of in vivo release of molt-inhibiting hormone and crustacean hyperglycemic hormone in the shore crab, Carcinus maenas.

Very little is known regarding the release patterns or circulating titers of neuropeptides in crustaceans, in particular those concerned with regulation of molting hormone (ecdysteroid) synthesis, molt-inhibiting hormone (MIH), and crustacean hyperglycemic hormone (CHH), which is also an adaptive hormone, centrally important in carbohydrate metabolism. Furthermore, the currently accepted model of molt control is founded on an untested hypothesis suggesting that molting can proceed only after decline in MIH titer. Accordingly, we measured simultaneous circulating neuropeptide profiles for both MIH and CHH by RIA of purified hemolymph during the molt cycle at fine temporal scale during day/night cycles and seasonally. For CHH we additionally determined release patterns after physiologically relevant stress. Results show that both hormones are released exclusively and episodically, rather than continuously, with notably short half-lives in circulation, suggesting dynamic and short-lived variations in levels of both hormones. During the molt cycle, there are no overt changes in MIH titer, except a massive and unprecedented increase in MIH during late premolt, just before ecdysis. The function of this hormone surge is unknown. Treatment with various stressors (hypoxia, temperature shock) showed that CHH release occurs extremely rapidly, within minutes of stress. Release of CHH after stressful episodes during premolt (when gut endocrine cells synthesize large quantities of CHH) is exclusively from the sinus gland: CHH from the gut is never involved in the stress response. The results show a hitherto unsuspected dynamism in release of MIH and CHH and suggest that currently accepted models of molt control must be reconsidered.

Characterization of cDNA encoding molt-inhibiting hormone of the crab, Cancer pagurus; expression of MIH in non-X-organ tissues.

Synthesis of ecdysteroids (molting hormones) by crustacean Y-organs is regulated by a neuropeptide, molt-inhibiting hormone (MIH), produced in eyestalk neural ganglia. We report here the molecular cloning of a cDNA encoding MIH of the edible crab, Cancer pagurus. Full-length MIH cDNA was obtained by using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides based upon the amino acid sequence of MIH, in conjunction with 5'- and 3'-RACE. Full-length clones of MIH cDNA were obtained that encoded a 35 amino acid putative signal peptide and the mature 78 amino acid peptide. Of various tissues examined by Northern blot analysis, the X-organ was the sole major site of expression of the MIH gene. However, a nested-PCR approach using non-degenerate MIH-specific primers indicated the presence of MIH transcripts in other tissues. Southern blot analysis indicated a simple gene arrangement with at least two copies of the MIH gene in the genome of C. pagurus. Additional Southern blotting experiments detected MIH-hybridizing bands in another Cancer species, Cancer antennarius and another crab species, Carcinus maenas.

Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630 Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1-40, but the remaining sequence, positions 41-73 or 41-72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH(41-76) sequence in the 3'-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH(1-40)/SG-CHH(1-40) sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3'-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules.

The Drosophila melanogaster homologue of an insect calcitonin-like diuretic hormone was identified in a BLAST search of the Drosophila genome database. The predicted 31-residue amidated peptide (D. melanogaster DH31; Drome-DH31) was synthesised and tested for activity on fruit fly Malpighian tubules. It increases tubule secretion by approximately 35 % of the response obtained with a myokinin from the housefly Musca domestica (muscakinin; Musdo-K) and has an EC50 of 4.3 nmol x l(-1). The diuretic activities of Drome-DH31 and Musdo-K were additive when tested at threshold and supra-maximal concentrations, which suggests that they target different transport processes. In support of this, Drome-DH31 increased the rate of secretion by tubules held in bathing fluid with a reduced Cl- concentration, whereas Musdo-K did so only in the presence of Drome-DH31. Stimulation with Drome-DH31 increased the lumen-positive transepithelial potential in the main secretory segment of the tubule. This was attributed to activation of an apical electrogenic proton-translocating V-ATPase in principal cells, since it was associated with hyperpolarisation of the apical membrane potential and acidification of secreted urine by 0.25 pH units. Exogenous 8-bromo-cyclic AMP and cyclic GMP increased tubule secretion to the same extent as Drome-DH31 and, when tested together with the diuretic peptide, their activities were not additive. Stimulation with Drome-DH31 resulted in a dose-dependent increase in cyclic AMP production by tubules incubated in saline containing 0.5 mmol x l(-1) 3-isobutyl-1-methylxanthine, whereas cyclic GMP production was unchanged. Taken together, the data are consistent with Drome-DH31 activating an apical membrane V-ATPase via cyclic AMP. Since the K+ concentration of the secreted urine was unchanged, it is likely that Drome-DH31 has an equal effect on K+ and Na+ entry across the basolateral membrane.

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon-intron boundary of the crab MIH and MO-IH genes follows Chambon's rule (GT-AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones.

Endocrine cells in the gut of the shore crab Carcinus maenas immunoreactive to crustacean hyperglycaemic hormone and its precursor-related peptide.

The distribution and morphology of gut endocrine cells, which are immunoreactive to crustacean hyperglycaemic hormone (CHH) and the corresponding precursor-related peptide (CPRP), have been described in the shore crab Carcinus maenas. The cells are uniquely distributed throughout the fore- and hindgut, but were never observed in the midgut or associated caeca. Expression of CHH and CPRP in the gut endocrine cells is generally restricted to premoult, although small numbers of immunoreactive cells were observed in intermoult and postmoult. A notable feature of the distribution of these slender cells was that, whilst they are distributed evenly over much of the fore- and hindgut, all extrinsic and intrinsic muscles of the gastric and pyloric stomach examined were surrounded by a ring(s) of cells, suggesting a mechanoreceptive function. Ultrastructural studies revealed that these cells contain numerous immunopositive, electron-dense granules. This suggests that they are "paraneurones", which secrete CHH and CPRP into the haemolymph during ecdysis, accounting for the ecdysial surge in CHH, which is implicated in water uptake and swelling prior to ecdysis.

Ecdysis of decapod crustaceans is associated with a dramatic release of crustacean cardioactive peptide into the haemolymph.

On the basis of detailed analyses of morphological characteristics and behavioural events associated with ecdysis in a crab (Carcinus maenas) and a crayfish (Orconectes limosus), a comprehensive substaging system has been introduced for the ecdysis stage of the moult cycle of these decapod crustaceans. In a remarkably similar stereotyped ecdysis sequence in both species, a passive phase of water uptake starting with bulging and rupture of thoracoabdominal exoskeletal junctions is followed by an active phase showing distinct behavioural changes involved in the shedding of the head appendages, abdomen and pereiopods. Together with an enzyme immunoassay for crustacean cardioactive peptide (CCAP), the substaging has been used to demonstrate a large, rapid and reproducible peak in haemolymph CCAP levels (increases of approximately 30-fold in the crab and more than 100-fold in the crayfish compared with intermoult titres) during the later stages of active ecdysis. We suggest that the release of CCAP (accumulated in late premoult) from the crab pericardial organs or the crayfish ventral nerve cord accounts for many of the changes in behaviour and physiology seen during ecdysis and that this neurohormone is likely to be of critical importance in crustaceans and other arthropods.

A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas.

Molting or ecdysis is the most fundamentally important process in arthropod life history, because shedding of the exoskeleton is an absolute prerequisite for growth and metamorphosis. Although the hormonal mechanisms driving ecdysis in insects have been studied extensively, nothing is known about these processes in crustaceans. During late premolt and during ecdysis in the crab Carcinus maenas, we observed a precise and reproducible surge in hemolymph hyperglycemic hormone (CHH) levels, which was over 100-fold greater than levels seen in intermolt animals. The source of this hormone surge was not from the eyestalk neurosecretory tissues but from previously undescribed endocrine cells (paraneurons), in defined areas of the foregut and hindgut. During premolt (the only time when CHH is expressed by these tissues), the gut is the largest endocrine tissue in the crab. The CHH surge, which is a result of an unusual, almost complete discharge of the contents of the gut endocrine cell, regulates water and ion uptake during molting, thus allowing the swelling necessary for successful ecdysis and the subsequent increase in size during postmolt. This study defines an endocrine brain/gut axis in the arthropods. We propose that the ionoregulatory process controlled by CHH may be common to arthropods, in that, for insects, a similar mechanism seems to be involved in antidiuresis. It also seems likely that a cascade of very precisely coordinated release of (neuro) hormones controls ecdysis.

Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus.

Methyl farnesoate, the crustacean juvenoid, is synthesized and secreted from the mandibular organs of crustaceans under the negative control of the sinus gland-derived mandibular organ-inhibiting hormone (MO-IH). Previously we isolated and sequenced two isoforms, MO-IH-1 and MO-IH-2, differing by just one amino acid, from sinus glands of the edible crab, Cancer pagurus. We now report the isolation of cDNAs encoding MO-IH-1 and MO-IH-2 by a combination of reverse-transcriptase-mediated PCR in conjunction with 5' and 3' rapid amplification of cDNA ends ('RACE'). Full-length clones of MO-IH-1 and MO-IH-2 encoded a 34-residue putative signal peptide and the mature 78-residue MO-IH sequences. Northern blot analysis of various tissues showed that MO-IH expression is confined to the X-organ (a cluster of perikarya within the eye). Southern blot analysis indicated that there are approx. 10 copies of the gene for MO-IH in C. pagurus. Additional Southern blotting experiments detected MO-IH-hybridizing bands in another Cancer species, C. antennarius. In support of this, an HPLC-radioimmunoassay analysis of sinus gland extracts of C. antennarius and C. magister also revealed MO-IH-like immunoreactivity.

Involvement of adenosine cyclic-3',5'-monophosphate in the signal transduction pathway of mandibular organ-inhibiting hormone of the edible crab, Cancer pagurus.

The juvenoid, methyl farnesoate (MF), is synthesized in the mandibular organs (MOs) of crustaceans, under the control of mandibular organ-inhibiting hormone (MO-IH). Using an in vitro assay to measure synthesis of MF by MOs, the effect of a variety of agents that affect signal transduction pathways was investigated. Of the compounds tested, only agents which affect cAMP (forskolin and 8-bromoadenosine cyclic-3',5'-monophosphate) levels were found to mimic the inhibitory action of MO-IH on MF synthesis. To further support these findings, the effect of MO-IH-1 on production of cAMP was investigated. The results demonstrated that MO-IH stimulated a dose-dependent increase in cAMP levels. Furthermore, a maximal 2-fold increase in cAMP was detected after a 5-min exposure of MO membranes to 100 nM MO-IH-1, falling to basal levels thereafter. The results presented strongly support a role for cAMP in the signal transduction mechanism of MO-IH that leads to inhibition of MF synthesis in MOs.

Amino acid sequences of both isoforms of crustacean hyperglycemic hormone (CHH) and corresponding precursor-related peptide in Cancer pagurus.

Both isoforms of the crustacean hyperglycemic hormone (CHH) and corresponding crustacean hyperglycemic hormone precursor-related peptide (CPRP) derived from HPLC-purified sinus gland extracts from the edible crab Cancer pagurus were fully characterised by microsequencing and mass spectrometry. The amino acid sequences of the CHH isoforms were almost identical except that the N-terminus of the minor isoform (CHH-I), was glutamine rather than pyroglutamate in the major isoform (CHH-II). Both CHH isoforms were of similar biological activity, as tested by in vivo hyperglycemia bioassays and in vitro repression of ecdysteroid synthesis. Comparison with other published CHH and CPRP sequences show that for crabs, these peptides form a distinct group, that the presence of CHH isoforms with free and blocked N-termini seems unique to crabs. It is argued that this phenomenon reflects a slow post-translational modification in sinus gland neurosecretory terminals. This study appears to complete the entire sinus gland inventory of functionally and structurally characterised CHH-related peptides in a crab.

Neuropeptide regulation of biosynthesis of the juvenoid, methyl farnesoate, in the edible crab, Cancer pagurus.

The neuropeptide mandibular organ (MO)-inhibiting hormone (MO-IH), synthesized and secreted from the X-organ-sinus-gland complex of the eyestalk, regulates the biosynthesis of the putative crustacean juvenile hormone, methyl farnesoate (MF). Using radiolabelled acetate as a precursor for isoprenoid biosynthesis, farnesoic acid (FA), farnesol, farnesal, MF and geranyl geraniol were detected in MOs cultured for 24 h. Treatment of MOs with extract of sinus gland inhibited the final step of biosynthesis of MF, catalysed by FA O-methyltransferase. Additionally, treatment of MOs with purified MO-IH exhibited a dose-dependent inhibition of this final step of MF synthesis. The extent of this inhibition was dependent on the ovary stage of the MO-donor animal, being maximal in MOs from animals in the early stages of ovarian development. Assay of FA O-methyltransferase activity, using [3H]FA in the presence of S-adenosyl-l-methionine, demonstrated that the enzyme was located in the cytosolic fraction of MOs and was inhibited by incubation of MOs with MO-IH prior to preparation of subcellular fractions. For cytosolic preparations taken from vitellogenic animals, both Vmax and Km were appreciably lower than for those taken from non-vitellogenic animals. Conversely, eyestalk ablation of early-vitellogenic animals, which removes the source of MO-IH in vivo, resulted in enhancement of the cytosolic FA O-methyltransferase activity. Although both Vmax and Km show an appreciable increase upon eyestalk ablation, the increased enzyme activity is probably reflected by the fact that Vmax/Km (an approximate indication of kcat) has increased 5-fold. The combined evidence demonstrates that MO-IH inhibits FA O-methyltransferase, the enzyme which catalyses the final step of MF biosynthesis in MOs.

Peptidergic Neurons in Barnacles: An Immunohistochemical Study Using Antisera Raised Against Crustacean Neuropeptides.

Antisera raised against neuropeptides from decapod crustaceans were used to investigate whether balanomorph barnacles produce peptides analogous to those identified in some decapods. The distribution and structure of immunoreactive neurons was examined in Balanus balanus, Balanus perforatus, and Chirona (Balanus) hameri by whole-mount immunohistochemistry. In these species, no immunoreactivity was observed to antisera against CHH (crustacean hyperglycemic hormone), MIH (molt-inhibiting hormone), or RPCH (red-pigment-concentrating hormone), but neurons immunoreactive for pigment-dispersing hormone (PDH) and crustacean cardioactive peptide (CCAP) were observed. In all three species, PDH immunoreactivity was primarily associated with a pair of large (30-50 {mu}m diam.) anterio-ventral perikarya in the ventral ganglion, projecting prominent axons along the great splanchnic nerves, which branched extensively in the segmental splanchnic nerves, directing several arborizing dendrites to the somatic extensor muscles. Occasionally, three pairs of anterio-dorsal perikarya were observed, which projected fine ipsilateral and contralateral axons along the great splanchnic nerves. A further 12 pairs of perikarya, apparently segmentally arranged, were observed in the thoracic ganglion. Several PDH-immunoreactive perikarya and associated branching plexus were observed in the supra-esophageal ganglion. CCAP immunoreactivity was mainly restricted to the ventral ganglion, where three pairs of perikarya (ca. 30-50 {mu}m diam.) projected contralateral descending axons to the cirri. Occasionally a single pair of immunoreactive neurons were observed in the supra-esophageal ganglia. Although the anatomy of the CCAP-immunoreactive neurons in the ventral ganglion of barnacles might be homologous to conserved neural architectures in higher crustaceans, the anatomy of the PDH-immunoreactive neurons seems unique, and the morphology of the two large neurons in the ventral ganglion suggests a neuromodulatory role for this peptide, possibly associated with somatic extension.

Does the N-terminal pyroglutamate residue have any physiological significance for crab hyperglycemic neuropeptides?

A characteristic feature of all crustacean hyperglycemic hormones (CHH) is that they are always present in the sinus gland as multiple forms or isoforms. The amino acid sequence of the minor form of CHH from the green shore crab, Carcinus maenas, was determined by automated microsequencing and MS, and was almost identical to that of the major form, except that the N-terminal residue was glutamine rather than pyroglutamate. Limited analysis (electrospray MS and amino acid composition) of the two corresponding forms of CHH from the edible crab, Cancer pagurus, suggested a similar phenomenon in this species. For C. maenas, both forms were indistinguishable in terms of their ability to cause sustained hyperglycemia in vivo and repression of ecdysteroid synthesis in vitro. Similarly, the two forms were immunologically identical in RIA, and exhibited similar binding characteristics in competitive-receptor-binding assays. CD studies showed only minor differences in presumed secondary structure. In vitro release experiments with isolated sinus glands demonstrated that both forms are probably released in a stoichiometric manner and that both peptides are present in the haemolymph at the same ratio as that in the sinus gland. Preliminary results suggest that the in vivo clearance/degradation rates of both peptides are similar. The unblocked (Gln) terminus is of particular significance, since the presence of this amino acid indicates that this peptide is derived from a precursor that does not possess the same structure of those of established preproCHH, or that N-terminal processing is slow, which results in the presence of unblocked CHH in sinus glands. The similar biological activity of the unblocked CHH to that of the blocked CHH suggests that the N-terminal pyroglutamate residue has no obvious biological significance (with respect to the known functions of CHH), an observation which is in contrast to the widely accepted paradigms concerning the stability and biological activity of N-terminally blocked and unblocked peptides.

Structure and significance of mandibular organ-inhibiting hormone in the crab, Cancer pagurus. Involvement in multihormonal regulation of growth and reproduction.

Current evidence indicates that methyl farnesoate is the crustacean equivalent of the juvenile hormones of insects. This putative hormone is produced by the mandibular organs and is negatively regulated by a neuropeptide produced and secreted by the X-organ-sinus gland complex of the eyestalk. To identify this neuropeptide, a bioassay was developed which measures the inhibition of methyl farnesoate synthesis by mandibular organs exposed to fractionated sinus gland extracts from the crab, Cancer pagurus. Two neuropeptides, named mandibular organ-inhibiting hormones (MOIH-1 and -2) repressed methyl farnesoate synthesis. MOIH-1 was fully sequenced by automated Edman degradation of endoproteinase-derived fragments and further characterized by mass spectrometry. This peptide consisted of 78 residues (Mr 9235.6), with unblocked termini and three intrachain disulfide bridges. MOIH-2 appeared to be almost identical to MOIH-1 with the exception of a Gln for Lys substitution at position 33. Comparison with previously sequenced crustacean neuropeptides shows that these MOIHs are members of the ever expanding crustacean hyperglycemic hormone family, with significant sequence similarity to molt-inhibiting hormones (MIHs). It is possible that these two structurally similar peptides (MIH, MOIH) may control mutually exclusive physiological phenomena (somatic and gonadal growth), suggesting a complex hormonal integration of these processes in crustaceans.

Determination of the amino acid sequence of the moult-inhibiting hormone from the edible crab, Cancer pagurus.

Putative moult-inhibiting hormone (MIH) from sinus glands of the edible crab Cancer pagurus was characterized by high-performance liquid chromatography, followed by fractional bioassay (inhibition of ecdysteroid synthesis by Y-organs) and immunoassay (using antisera raised against Carcinus MIH). This peptide was fully sequenced by automated Edman degradation of endoproteinase-derived fragments. C. pagurus MIH is a 78 residue peptide (M(r) 9194), with free N- and C-termini and three intrachain disulphide bridges. Comparison with previously published MIH sequences confirms a high degree of sequence identity (c. 80%), supporting the view that brachyurans (crabs), possess distinct, structurally similar MIH neuropeptides.