S. Geoffre

Immunoreactive gonadotropin-releasing hormone-like material in the brain and the pituitary gland during the periovulatory period in the brown trout (Salmo trutta L.): Relationships with the plasma and pituitary gonadotropin

In fish there are few data on the gonadotropin-releasing hormone (Gn-RH) neurosecretory activity, which could explain long- and short-term variations of the gonadotropin secretion. There is no biological species specificity between mammal and fish Gn-RH; although there is a structural difference, they are, on the contrary, characterized by a high immunological specificity which does not allow measurement of fish Gn-RH using radioimmunoassay for LH-RH. We have synthesized salmon Gn-RH according to the formula recently proposed by Sherwood (N. Sherwood, L. Eiden, M. Brownstein, J. Spies, J. Rivier, and W. Vale, 1983. Proc. Natl. Acad. Sci. USA 80, 2794-2798). Its activity has been tested by its ability to stimulate the gonadotropin hormone (GtH) secretion in vivo in testosterone-implanted juvenile rainbow trout, and for the recognition of synthesized Gn-RH (s-Gn-RH) perykaria by a specific antibody raised against the s-Gn-RH in regions of the brain described as containing LH-RH immunoreactive-like material. A radioimmunoassay has been developed for the salmon Gn-RH, and its specificity to measure trout Gn-RH has been tested. Using this assay, the brain and pituitary Gn-RH contents have been measured throughout the final phases of maturation and ovulation. Brain Gn-RH increases from the end of vitellogenesis (8.9 +/- 0.76 ng/brain) to ovulation (more than 15 ng/brain). Pituitary Gn-RH is lower (1.58 +/- 0.69 ng/pituitary) at the end of vitellogenesis and follows a similar profile as in the brain, except for a significant decrease just prior the beginning of oocyte maturation. The correlations between Gn-RH levels and GtH pituitary and plasma levels show that total brain Gn-RH is never correlated to the GtH, suggesting that the increase in the brain Gn-RH content is related to a Gn-RH system closely related to maturation and ovulation, which remains to be investigated. On the contrary, pituitary Gn-RH levels are well correlated with pituitary and plasma GtH levels, indicating that pituitary Gn-RH levels might represent a good index of the Gn-RH neurosecretory activity in the fish hypothalamohypophysial complex, given the absence of a portal system in teleost.

Arguments for two distinct gonadotropic activities triggered by different domains of the ovary maturating parsin of Locusta migratoria

To complete previous results concerning the role of the ovary maturating parsin of Locusta migratoria (Lom OMP), we determined, by an enzyme immunoassay, the titers of circulating ecdysteroids and analyzed circulating vitellogenin (Vg) and oöcyte growth following (1) suppression of 20 hydroxyecdysone (20E) and (2) injection of the Lom OMP, either as an entire molecule in allatectomized adults or as smaller peptides in allatectomized fifth-instar larvae females. Titers of ecdysteroids appeared unrelated to the presence of circulating Vg but increased during the first phase of vitellogenesis and injection of OMP accelerated the occurrence of circulating 20E. Nevertheless, immunoneutralization of 20E at the beginning of adult life delayed but did not prevent rapid oöcyte growth contrary to immunoneutralization of Lom OMP suggesting an additive gonadotropic effect of the neurohormone, distinct from that of 20E. Of two synthetic peptides corresponding to the C- and N-terminal gonadotropic domains of the OMP, respectively, only the C-terminal peptide was able to induce Vg in allatectomized larvae. After metamorphosis, injection of OMP did not induce Vg in adults allatectomized at the beginning of imaginal life but improved the maintenance of circulating Vg in adults allatectomized after Vg appeared in the haemolymph. This result suggests that OMP either delays the Vg mRNA decay or increases the translation of Vg mRNA. Thus, Lom OMP appears to have two distinct roles: an ecdysteroidogenic effect triggered by its C-terminal domain with the ovary as the target tissue and a protecting effect on Vg mRNA probably triggered by its other gonadotropic domain, the N-terminal, with the fat body as the target tissue.

Covalent modification of a melanoma-derived antigenic peptide with a natural quinone methide. Preliminary chemical, molecular modelling and immunological evaluation studies

A LigandFit shape-directed docking methodology was used to identify the best position at which the melanoma-derived MHC class-I HLA-A2-binding antigenic peptide ELAGIGILTV could be modified by attaching a small molecule capable of fitting at the interface of complementary determining regional (CDR) loops of a T-cell receptor (TCR) while triggering T-cell responses. The small molecule selected here for determining the feasibility of this alternative track to chemical alteration of antigenic peptides was the electrophilic quinone methide (+)-puupehenone (), a natural product that belongs to a family of marine metabolites capable of expressing immunomodulatory activities. A preliminary chemical reactivity model study revealed the efficacy of the thiol group of a cysteine (C) side-chain in its nucleophilic addition reaction with in a regio- and diastereoselective manner. The best TCR/HLA-A2 ligand [i.e., ELAGCGILTV-S-puupehenol ()] then identified by the LigandFit docking procedure was synthesized and used to pulse HLA-A2(+) T2 cells for T-cell stimulation. Among the ELAGIGILTV-specific T-cell clones we tested, five of them recognized the conjugate in spite of its low binding affinity for the HLA-A2 molecules. The resulting T-cell stimulation was determined through the intracytoplasmic secretion of IFN-gamma and the percentage of T-cells thus activated. These highly encouraging results indicate that small non-peptidic natural product-derived molecules attached onto the central part of an antigenic peptide can fit at the TCR/HLA-A2 interface with induction of T-cell responses.

Synthesis and CD studies of an 88-residue peptide containing the main receptor binding site of HTLV-I SU-glycoprotein.

Essential HTLV‐I biological functions, like host‐cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg147‐Leu234] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid‐phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non‐random conformation of at least 70–80% of the synthetic peptide. Investigation of the 3D‐structure of the synthetic peptide will provide useful information for future vaccine and drug‐design strategies.

Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide–resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane‐embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt α‐helical secondary structure is evidenced by circular dichroism spectroscopy. Copyright

X-RAY CRYSTALLOGRAPHY OF LINEAR PEPTIDES THROUGH THE RENIN–ANGIOTENSIN SYSTEM

ABSTRACT A-ray crystallographic studies have been done on inhibitors and substate fragments of renin, The preliminary results show a good geometrical analogy between the crystal conformation of a pepstatin analogue and the conformation observed on the site of an acidic proteinase. The structure solutions of many different molecules allow to point out some characteristics always found in the conformations of peptidic fragments implicated in the renin angiotensin reaction.