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Eoexechia gallica gen. n. and sp. n., oldest record of the fungus gnat tribe Exechiini, is described from the Lowermost Eocene amber of France. It falls as sister group of all the extant Exechiini after its addition to the morphological phylogenetic analysis of the tribe. Its discovery suggests a Paleocene age for the Exechiini, in accordance with the current phylogenetic dating. This new discovery will help to date the whole clade in future phylogenies.

Bovine corpus luteum is the site of intense production of pro-ocytocin-neurophysin mRNA at day 1 after estrus (Ivell et al. (1985) FEBS Lett. 190, 263-267) which is followed by apparent delayed production of ocytocin. Therefore it is a good model to study both the translational and post-translational production of this neuropeptide in non-hypothalamic tissues and its regulation. In order to assess if this mRNA is translated during the lag period we have analyzed the neurophysin-like species produced in this organ. As early as day 2 after estrus one neurophysin species (pI approximately 4.7) could be detected and was unequivocally identified as pro-ocytocin-neurophysin. In primary cultures of luteinizing granulosa cells, biosynthetic intermediates were characterized, i.e. ocytocin-Gly, ocytocin-Gly-Lys and ocytocin-Gly-Lys-Arg, whereas amidated, fully mature, ocytocin was undetectable. We conclude that translation of pro-ocytocin-neurophysin mRNA takes place soon after transcription and we propose that incomplete processing could be responsible for the low level of ocytocin in the early bovine corpus luteum.

The current knowledge on the biosynthetic mechanisms of three hypothalamic neuropeptides, neurophysins, vasopressin and somatostatin, is reviewed. Both neurophysin and vasopressin appear to be first synthesized as higher molecular weight precursors. The methodology elaborated allows to characterize essentially two main forms with apparent Mr similar to or approximately 25 000 and 80 000 respectively. It can be demonstrated that both neurophysin and vasopressin are derived from common precursors.

Ocytocin and vasopressin are two nonapeptides of the hypo thalamo-Post-hypophysary system. They are isolated and purified from dromedary post-hyphophysis, freshly collected in the meat centers of the south of Tunisia. The use of three chromatographies steps were found to be essential in obtaining high Pure hormones. These steps cousist in a sephadex G25 gel filtration and two successive High performance liquid chromatographies: H.P.L.C. The analysis of the two hormones by thin layer chromatography and Radioimmunoassays, and the identification of the amino-acid composition demonstrate that camel vasopressin is a 8 arginin vasopressin but ocytocin is identic to the other hormones isolated from different animal species.

Purified secretory granules from the corpus luteum of super ovulated and fecundated cows, at day 7-8 after the heat period, were used as a source of pro-ocytocin/neurophysin (pro OT/Np) processing enzymes. An endoprotease comparable to the previously described pituitary enzyme both by its catalytic properties and sensitivity to various inhibitors was characterized. This protease cleaves pro OT/Np (1-20) after the basic Lys11Arg12 doublet to release OT Gly10 Lys11 Arg12. Moreover C-terminally extended ocytocin, i.e. OTGlyLys and OTGlyLysArg together with ocytocin were identified in extracts from the corpus luteum. Together these data argue strongly in favor of pro OT/Np processing pathways in which cleavage of the precursor at the Arg12-Ala13 peptide bond is the primary event.

Purified bovine neurosecretory granules and their corresponding membranes were prepared after fractionation and purification processes from bovine pituitaries. An adenylate cyclase activity was detected both in the granules (apparent Km = 0.5 mM) and the corresponding preparations of the membranes (apparent Km = 0.5 mM). This enzyme was activated by fluoride in a way markedly dependent on the concentration of this ion, and with a maximum for a concentration of F- = 3.5 mM. The cyclase activity was also significantly enhanced by GTP. The reaction rate showed a strong dependence on the molar ration [Mg2+]/[ATP] with maximal velocity for 7. It is suggested that this activity might play an important role in the control and regulation of neurosecretion in the neurohypophysis.

The in vivo incorporation of [3H]phenylalanine into an apparent 15 kDa prosomatostatin was observed in the hypothalamus of rats injected with the labeled amino acid in the third ventricle. Precursor-product relationships were established between this newly synthesized material and both somatostatin-28 and -14.

We have tested the hypothesis that the high M(r) forms common to both neurophysin and vasopressin detected in bovine neurohypophysis extracts (Nicolas, P., Camier, M., Lauber, M., Masse, M.-J. O., Möhring, J. & Cohen, P. (1980) Proc. Natl. Acad. Sci. USA 77, 2587-2591) might also contain the sequences of other known neuropeptides. The following evidence indicates that corticotropin- and beta-endorphin-like sequences are associated with similar high M(r) forms and are included in these M(r) 80,000 molecules. During the fractionation steps of high M(r) material, both corticotropin and beta-endorphin immunoreactive species were found to coelute with the neurophysin and vasopressin ones, either under M(r) 140,000 (in 0.1 M formic acid) or M(r) 70,000-80,000 (in 6 M guanidine) elution volumes. Corticotropin immunoreactivity was found to cofocus at pIs 6.05 and 5.8 with the M(r) 80,000 neurophysin-containing species. This material was submitted to affinity chromatography on purified anti-neurophysin antibodies covalently attached to Sepharose 4B. Both the corticotropin and beta-endorphin immunoreactivities, together with the neurophysin and vasopressin immunoreactivities, were retained on the immunoadsorbent and codesorbed by either a drastic pH change or by selective displacement with an excess of neurophysin. Comparison of the tryptic-digest maps of either the M(r) 68,000 fragment immunoprecipitated by anti-corticotropin antibodies or the M(r) 68,000 fragment released after precipitation of the M(r) 80,000 species by anti-neurophysin antibodies indicated large sequence homologies. Exposure of either the M(r) 80,000 or 68,000 components to mild proteolytic activities resulted in the formation of lower-size fragments. The resulting corticotropin-like immunoreactive material, recovered under the elution volume of standard (125)I-labeled corticotropin-(1-24), was tested for its ability to activate glucocorticoid biogenesis by the amphibian interrenal tissue (adrenal) in perifusion. It was found to exhibit a noticeable activity qualitatively undistinguishable from the one of the reference human corticotropin-(1-39). The name neurohypophyseal "coenophorin" (from the Greek word for common) is proposed for this class of M(r) 80,000 polypeptides that might represent the common precursor store-house for a set of neuropeptides produced in the hypothalamo-neurohypophyseal tract.

Proton magnetic resonance spectroscopy was used to monitor individual amino acid residues in bovine neurophysin, in the nonapeptide hormone oxytocin, and in the complex formed between them. For neurophysin I alone, a normal titration curve for the C-2 proton resonance of the lone histidine residue was obtained with an apparent ionization constant of 6.9 addition of oxytocin to a solution of neurophysin I at pH 6.5 resulted in several changes in the spectrum. The effect on the histidine C-2 proton resonance signal indicated a slow exchange process between two states, probably representing a conformational change in the protein. The apparent pK of the histidine residue in the hormonal complex was shifted to 6.7, indicating a slightly more positive (less electron dense) environment for the histidine residue. Resonances of the single tyrosine residue of oxytocin were observed to broaden significantly, but not to shift appreciably, on the addition of neurophysin II. These observations may indicate involvement of the tyrosyl residue of oxytocin in the hormone-"carrier protein" interaction.

The octacosapeptide sequence [Tyr18] pro-ocytocin/neurophysin (1-18)NH2 [pro-OT/Np(1-18)NH2] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in Lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly10-Lys11-Arg12+Ala13-Val-Leu-Asp-Leu-Tyr18 (NH2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly10-Lys11-Arg12 into OT-Gly10 was detected in the same granule Lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT/Np in vivo.

Twenty-four hours after the injection of [35S]cysteine near either the rat paraventricular nuclei or the supraoptic nuclei, the [35S]neurophysin-like proteins of the brain stem were extracted, immunoprecipitated with anti-bovine neurophysins antibodies and analyzed. They consisted essentially of species behaving as neurophysin on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. There was a very low percentage of neurophysins precursors which could be characterized in the paraventricular nuclei. In the rats pretreated by colchicine, the [35S]neurophysins were not detected in the brain stem, while they appeared in the paraventricular nuclei indicating that the precursors have been processed and the transport inhibited. These results suggest that: (i) both the biosynthetic and transport events in the hypothalamo-brain stem pathway are comparable to those occurring in the hypothalamo-neurohypophyseal tract; (ii) this pathway originates both from the supraoptic and paraventricular nuclei. Moreover, they indicate that processing is essentially complete in the hypothalamus of colchicine-pretreated animals. This provides further support to a model associating enzymes with both the endoplasmic reticulum membranes and the derived corresponding secretory vesicles.

Extracts of mouse hypothalamus made in acid/urea containing protease inhibitors were analyzed for somatostatin immunoreactivity after molecular sieve filtration on Sephadex G-50. Higher molecular weight (higher-M(r)) somatostatin-like forms with apparent molecular weights of 15,000, 10,000, and 6000 could be identified, besides the molecular weight 1600 somatostatin. Immunological identities with somatostatin were unambiguously demonstrated by the analysis of the displacement curves in the radioimmunoassay. The M(r) 15,000, 6000, and 1600 species were purified by affinity chromatography on an anti-somatostatin immune serum covalent conjugate with Sepharose used as immunoadsorbant. After disulfide reduction by dithiothreitol, the size of the M(r) 15,000 and 6000 somatostatin-like species was assessed either by molecular sieve filtration or by polyacrylamide gel electrophoresis. The results indicated that the higher-M(r) somatostatin-like species isolated from the hypothalamus did not result from hormone polymerization by means of disulfide interchange. The processing in vitro of the 15,000 higher-M(r) form of somatostatin was achieved by proteolytic enzymes coeluted with this species during the fractionation of hypothalamic extracts. Under neutral pH conditions the intermediary higher-M(r) forms were generated together with the M(r) 1600 somatostatin-like species. This processing activity could be either strongly inhibited at acidic pH or in acid/urea medium or else eliminated by selective immunoadsorption of the 15,000 higher-M(r) form. Neither trypsin nor the gamma subunit of 7S nerve growth factor was able to produce this processing, suggesting that enzymes with other kinds of specificity may be involved. It is concluded that somatostatin biosynthesis in the mouse hypothalamus may occur via a high-M(r) precursor that is processed into intermediary forms leading to the tetradecapeptide hormone.