Search

The insulin-like androgenic gland hormone (IAG) found in decapod crustaceans is known to regulate sexual development in males. IAG is produced in the male-specific endocrine tissue, the androgenic gland (AG); however, IAG expression has been also observed in other tissues of decapod crustacean species including Callinectes sapidus and Scylla paramamosain. This study aimed to isolate the full-length cDNA sequence of IAG from the AG of male red deep-sea crabs, Chaceon quinquedens (ChqIAG), and to examine its tissue distribution. To this end, we employed polymerase chain reaction cloning with degenerate primers and 5′ and 3′ rapid amplification of cDNA ends (RACE). The full-length ChqIAG cDNA sequence (1555 nt) includes a 366 nt 5′ untranslated region a 453 nt open reading frame encoding 151 amino acids, and a relatively long 3′ UTR of 733 nt. The ORF consists of a 19 aa signal peptide, 32 aa B chain, 56 aa C chain, and 44 aa A chain. The putative ChqIAG amino acid sequence is most similar to those found in other crab species, including C. sapidus and S. paramamosain, which are clustered together phylogenetically.

The blue crab Callinectes sapidus reovirus 1 (CsRV1), a double-stranded RNA virus, belongs to the Class III virus family Reoviridae and infects marine invertebrates, including the blue crab C. sapidus. Callinectes sapidus reovirus 1 levels correlate with disease progression, and high levels of infection usually result in death. To understand CsRV1 disease progression, the impact of CsRV1 infection on the translation initiation machinery in adult female C. sapidus was determined. Using transcriptome analysis of C. sapidus, sequences representing each of the three classes of metazoan eukaryotic translation initiation factor 4E (eIF4E) family members, eIF4E1, eIF4E2, and eIF4E3, as well as the eIF4E-binding protein (4E-BP), were identified as the first description of a Class III eIF4E in decapod crustaceans. Transcripts encoding the three eIF4E family members and 4E-BP are found in all tissues examined by end-point RT-PCR analysis, except for the hypodermis. In the noninfected animals, EIF4E1 transcript levels are;11.5- to 150-fold higher than those of EIF4E2, EIF4E3, or 4EBP in both hemocytes and eyestalk ganglia. In CsRV1 infected animals, transcript levels of eIF4E family members and 4EBP initially increase in hemocytes and to a much lesser extent in eyestalk ganglia. In hemocytes, EIF4E2, EIF4E3, and 4EBP transcript levels are highest at low levels of infection. By contrast, EIF4E1 transcript levels peak at moderate levels of infection. In the eyestalk ganglia, an endocrine tissue, CsRV1 infection only slightly impacts EIF4E1 and EIF4E2 levels, although more substantial increases in EIF4E3 and 4EBP are seen. These results provide the first window into how CsRV1 infection and disease progression perturb the host C. sapidus eIF4E family members and 4E-BP in different tissues, suggesting nuanced responses of the host translationalmachinery during disease progression.

Background: Neuropeptides are examples of small, flexible molecules that bind to receptors and induce signal transduction, thereby eliciting biological activity. The multifunctional insect kinin neuropeptides retain full activity when reduced to only their carboxy-terminal pentapeptide (Phe 1 -X 2 -X 3 -Trp 4 -Gly 5 -NH 2 ), thereby allowing extensive structure-function studies and conformational analysis. Results: A combined experimental and theoretical analysis of the insect kinin carboxy-terminal pentapeptide was used to probe the role of each residue, define the bioactive conformation, and design a constrained bioactive analog. Coupling receptor-binding data with two biological activity assays allowed receptor binding and signal transduction to be differentiated. A preferred β-turn conformation, found for residues 1–4 by molecular dynamics simulations, was tested by designing a conformationally restricted cyclic hexapeptide. This cyclic analog showed a preference for the β-turn conformation, as shown by a conformational search and nuclear magnetic resonance spectroscopy, and it showed stronger receptor binding but decreased activity relative to highly active linear analogs. Conclusions: Each residue of the insect kinin carboxy-terminal pentapeptide has a distinct role in conformational preference, specific receptor interactions or signal transduction. The (β-turn preference of residues Phe 1 -X 2 -X 3 -Trp 4 implicates this as the bioactive conformation. The amidated carboxyl terminus, required for activity in many neuropeptide families, may be generally important for signal transduction and its inclusion may therefore be essential for agonist design.

Achetakinin-like immunoreactive material in tissues and haemolymph of adult male crickets was quantified by radioimmunoassay. Achetakinin-like material was found in the brain, suboesophageal ganglia and the thoracic and abdominal ganglia, but the largest amount was within the retrocerebral complex. A Ca2+-dependent release of achetakinin-like immunoreactive material was demonstrated from retrocerebral complexes incubated in vitro in saline containing a high concentration of K+. The concentration of achetakinin-like material in haemolymph from fed crickets was estimated to be 2.8 nmol l−1 and increased more than 10-fold in insects starved for 48 h without access to water. The presence of achetakinin-like material in haemolymph suggests that these peptides are released in vivo and function as circulating neurohormones.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

Previously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-duca-DH) has been isolated from Manduca sexta and shown to stimulate fluid excretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering postfeeding Manduca sexta larvae. However, Manduca-DH was reported to have no effect on Malpighian tubules in vitro. Manduca-DH and [Nle2,11]-Manduca-DH were synthesized in Texas and assayed in London on isolated Malpighian tubules of Acheta domesticus. Man-duca-DH stimulated fluid secretion by about 60% of the maximum response achievable with extracts of corpora cardiaca and increased the production of cyclic AMP. In combination with 10−4moll−1 3-isobutyl-l-methyl xanthine (IBMX), Manduca-DH stimulated maximal secretion. A number of CRF-related peptides also stimulated fluid secretion and cyclic AMP production in cricket tubules, and the CRF antagonist α-helical-CRF[9–41] blocked the stimulation of fluid secretion by Manduca-DH. [Nle2,11]-Manduca-DH was more active than Manduca-DH in both assays, suggesting that methionine residues in the natural peptide may become oxidized. Taken in conjunction with previous in vivo studies, the present findings suggest that a Manduca-DH-like diuretic peptide is the hormone controlling post-eclosion diuresis in butterflies, and Manduca-DH was shown to stimulate both fluid secretion and cyclic AMP production in Malpighian tubules from 1–12 h posteclosion Pieris rapae adults. The function of the peptide in Manduca sexta is discussed.

A biologically active 125 I-labeled analogue of AK-II (3′-hydroxyphenyl propionic-Gly-Gly-Gly-Phe-Ser-Pro-Trp-Gly-NH 2 ) was used to investigate the properties of achetakinin binding sites on plasma membranes from Malpighian tubules of Acheta domesticus . With optimized conditions, binding was rapid, reversible, and specific, and saturation studies revealed a single class of binding sites with K d 0.55 n M and B max 39.9 fmol/mg membrane protein. The affinities of achetakinins for binding sites on tubule membranes ranked AK-V > AK III > AK-II > AK-I ≥ AK-IV, in general agreement with their potencies in functional assays. However, IC 50 values were several orders of magnitude higher than corresponding values for EC 50 , which suggests a considerable receptor reserve.