RP-HPLC profile of the crude extract of sinus glands.

<p>Mobile Phase A: 0.1% TFA in water. Mobile Phase B: 0.1% TFA in acetonitrile. Gradient: 0–100% B over 60 min at 1 mL min⁻¹. Column: Zorbax SB-C18 4.6 × 150 mm.</p

Horizontal time sequence of experimental design.

<p>Horizontal time sequence of experimental design.</p

Mean time spent motionless (+SE) in Control Pairs (CP), Reinforced Pairs (RP) and Inverted Pairs (IP), and in alphas (white bars) and betas (black bars).

<p>One and three asterisks denote significant differences at P<0.05 and P<0.001, respectively, after Student’s t-tests.</p

Behavioural parameters across fighting bouts (T0, T1, T2, T3) in Control Pairs (CP), Reinforced Pairs (RP), and Inverted Pairs (IP).

<p>Before T0, the initial glycemia was determined; during T0, alpha and beta crayfish were assessed; between T0 and T1, crayfish were subject to the injection of either PBS solution (both alphas and betas in CP, alphas in IP, and betas in RP) or cHH solution (betas in IP and alphas in RP); from T1 to T3, crayfish behaviour was recorded and, then, the final glycemia was determined. Means (± SE) of: (a) duration of fights; (b) percentage of dominance; (c) fight intensity level; (d) number of fights started by alphas. One and two asterisks denote significant difference at P<0.05 and P<0.01, respectively, after one-way ANOVAs.</p

Scheme of the SPPS coupled to NCL to obtain the full length peptides.

<p>The leading segments containing a C-terminal thioester, QVF-cHH4-38, QVdF-cHH4-38, pEVdF-cHH4-38, and the cHH39-72 following segment with a free N-terminal cysteine were synthesized by SPPS. The NCL reaction between the leading and following segments returned the full length 72mer cHH isomers, which were further subjected to oxidative folding to obtain the folded peptides.</p

Boxplots of the maximum glucose values recorded for the synthetic and native peptides.

<p>Both D-cHH and Glp-D-cHH induced a strong hyperglycemia with a mean peak glucose concentration of, respectively, 100.5±9.3 and 98.7±6 mg/dL, while the hyperglycemic effect of GS-cHH and of L-cHH was lower, with a mean of max glycemia of 52.7±9.4 mg/dL and of 52.4±6.4 mg/dL respectively.</p

Time course of the induced glycemia after injection of the synthetic and wild type cHHs.

<p>a) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of D-cHH. The maximum peak of 95.4±8.1 mg/dL glucose is reached after 4 h, high values lasting at 8 h (76±12.1 mg/dL), when the maximum values were also recorded, and the glycemia returning to its nearby basal level, 8.9±1 mg/dL, after 24 h. N = 20. b) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of L-cHH. L-cHH induced a slower hyperglycemic response with the maximum peak of 52±6.5 mg/dL glucose after 2 h, and the glycemia returning to its nearby basal level, 5.5±0.4 mg/dL, after 8 h. N = 16. c) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of Glp-D-cHH. The time course of Glp-D-cHH shows the stronger hyperglycemic response of 96.1±5.6 mg/dL glucose after 4 h, high values lasting at 8 h (62.9±11.8 mg/dL) and the glycemia returning to its basal level, 2.9±0.5 mg/dL, after 24 h. N = 9. d) Time course of hemolymph glycemia after injection of 1.7 pmol/g live weight of SG-cHH. The hyperglycemic time course after the injection of 1.7 pmol/g live weight of purified SG-cHH was comparable to that of synthetic D-cHH and Glp-D-cHH peptides, but the maximum (44±4.5 mg/dL) was reached after 2 h and the glycemia returning to its basal level, 5±1.1 mg/dL after 24 h. N = 9.</p

RP-HPLC profiles and ESI-MS spectra of the synthesized cHH isomers.

<p>Reverse Phase HPLC profiles of the purified refolded synthetic polypeptides and their corresponding ESI-MS multiply charged ion and deconvoluted spectra (insets): L-Phe³-cHH (A and B), D-Phe³-cHH (C and D), Glp-D-Phe³-cHH (E and F). Mobile Phase A: 0.1% TFA in water. Mobile Phase B: 0.1% TFA in MeCN. Gradient: 0–100% B over 25 min. Column: Gemini 5 C18 2.0×150 mm.</p