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International audience; To assess functional importance of the residues in the amino-and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.

Background Crustacean Hyperglycemic Hormone (CHH) family peptides are neurohormones known to regulate several important functions in decapod crustaceans such as ionic and energetic metabolism, molting and reproduction. The structural conservation of these peptides, together with the variety of functions they display, led us to investigate their evolutionary history. CHH family peptides exist in insects (Ion Transport Peptides) and may be present in all ecdysozoans as well. In order to extend the evolutionary study to the entire family, CHH family peptides were thus searched in taxa outside decapods, where they have been, to date, poorly investigated. Results CHH family peptides were characterized by molecular cloning in a branchiopod crustacean, Daphnia magna, and in a collembolan, Folsomia candida. Genes encoding such peptides were also rebuilt in silico from genomic sequences of another branchiopod, a chelicerate and two nematodes. These sequences were included in updated datasets to build phylogenies of the CHH family in pancrustaceans. These phylogenies suggest that peptides found in Branchiopoda and Collembola are more closely related to insect ITPs than to crustacean CHHs. Datasets were also used to support a phylogenetic hypothesis about pancrustacean relationships, which, in addition to gene structures, allowed us to propose two evolutionary scenarios of this multigenic family in ecdysozoans. Conclusions Evolutionary scenarios suggest that CHH family genes of ecdysozoans originate from an ancestral two-exon gene, and genes of arthropods from a three-exon one. In malacostracans, the evolution of the CHH family has involved several duplication, insertion or deletion events, leading to neuropeptides with a wide variety of functions, as observed in decapods. This family could thus constitute a promising model to investigate the links between gene duplications and functional divergence.

International audience; On the basis of formerly investigated peptides corresponding to the endotoxin-binding domain from the Limulus anti-lipopolysaccharide factor (LALF), a protein from L. polyphemus, we have designed and synthesized peptides of different lengths with the aim to get potential therapeutic agents against the septic shock syndrom. For an understanding of the mechanisms of action, we performed a profound physico-chemical and biophysical analysis of the interaction of rough mutant lipopolysaccharide (LPS) with these peptides by applying Fourier-transform infrared spectroscopy (FTIR), small-angle X-ray scattering (SAXS), calorimetric techniques (differential scanning calorimetry DSC and isothermal titration calorimetry ITC), and freeze-fracture transmission electron microscopy (FFTEM). Also, the action of the peptides on bacteria of different origin in microbial assays was investigated. Using FTIR and DSC, our data indicated a strong fluidization of the lipid A acyl chains due to peptide binding, with a decrease of the endothermic melting enthalpy change of the acyl chains down to a complete disappearance in the 1:0.5 to 1:2 [LPS]:[peptide] molar ratio range. Via ITC, it was deduced that the binding is a clearly exothermic process which runs into saturation also at a [LPS]:[peptide] = 1:0.5 to 1:2 molar ratio. The data obtained with SAXS indicated a drastic change of the aggregate structures of LPS into a multilamellar stack, which was visualized in electron micrographs as hundreds of lamellar layers. This can be directly correlated with the inhibition of the LPS-induced production of tumor-necrosis-factor-α in human mononuclear cells, but not with the action of the peptides on bacteria.

A tissue culture approach was used to monitor changes in protein secretion by integuments from Apis mellifera along the pupal stage. A dramatic change in epidermal protein secretion was correlated with the variation in endogenous ecdysteroid from a low level at the beginning of the pupal stage to a maximum level in the middle of this stage. Only a fraction of these proteins, however, turned out to be directly regulated by ecdysteroids when we exposed cultured integument to 20-hydroxyecdysone. An antiserum raised against a honey bee activated prophenoloxidase (proPO) found in hemolymph recognized a 70 kDa subunit in pupal integument incubations, strongly suggesting that honey bee epidermis synthesizes a genuine proPO. The 70 kDa subunit is not developmentally regulated by the ecdysteroid titer. Its constitutive expression throughout the pupal stage makes it difficult to reconcile the function of this protein with progressive cuticle pigmentation in late honey bee pupae. Apis mellifera / integument / epidermis / cuticle / cuticular protein / prophenoloxidase

L'évolution du taux de gonadotropines a été suivie dans le plasma par dosage radioimmunologique chez Carassius auratus au cours du nycthémère et pendant la période d'ovulation. Les résultats mettent en évidence:1. L'existence d'une forte décharge hormonale dans le sang au moment de l'ovulation (Fig. 5).2. Une évolution cyclique du contenu hormonal plasmatique au cours du nyethémère particulièrement nette chez les femelles (Fig. 3), avec un maximum à 11 heures du matin dans le nyethémère de juillet.Abstract : Changes in plasmatic gonadotropin level were observed by radioimmunologie assay in Carassins auratus for a 24-hr period during ovulation. The following results were found:A high hormone discharge into the blood at the time of ovulation (Fig. 5).A particularly distinet change of plasmatic hormone content over a 24-hr period in the females (Fig. 3) which is at its maximum at 11 am in the 24-hr period of July.