Partial characterization of a novel cardioinhibitory peptide from the brain of the snail Helix aspersa.

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin. 2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure: H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH2. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-amino acid sequence of HCIP is identical to that of FMRFamide family peptides. 3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified.

Cross-reactivity between terrestrial snails (Helix species) and house-dust mite (Dermatophagoides pteronyssinus). II. In vitro study.

Epidemiologic and in vitro data have shown that the association of house-dust mite (HDM) allergy and snail allergy in the same patients was due to cross-reactivity between HDM and snail allergenic components. However, the cross-reacting allergen(s) have not yet been identified. In vitro reactivity of seven patients' sera to the various extracts and hemolymph of four different Helix snail species was analyzed by IgE detection and immunodots and Western blots. Cross-reactivity between snails and Dermatophagoides pteronyssinus was assessed by immunodot and ELISA inhibition in two patients. Heterologous inhibition of the snail immunodot and ELISA was observed in one serum. Western blotting showed a specific binding on all four snail species extracts; molecular weights of snail allergens ranged from < 21 to 200 kDa. Marked individual differences were observed in the seven sera under study; most sera demonstrated IgE recognition of multiple bands, illustrating that no single allergen is responsible for cross-reactivity between snail and mite. These results confirm that cross-reactivity exists between snails of the Helix genus and HDM. This cross-reactivity, involving more than a single allergen, may be of clinical significance in atopic patients allergic to D. pteronyssinus. The identity of the cross-reacting allergens remains to be determined. Potential candidates include the thermostable minor allergens of D. pteronyssinus, tropomyosin and hemocyanin.

[In vivo and in vitro study of the effect of somatostatin and somatostatin-immunoreactive substance on the histology of the mantle edge in adult and juvenile snails (Helix aspersa)]. / Etude in vivo et in vitro de l'effect de la somatostatine et d'une substance somatostatine-immunoréactive sur l'histologie du manteau d'escargots (Helix aspersa) adultes et juvéniles.

We have demonstrated structural differences between the mantle edge gland of young, adult and adult shell repairing snails. In vivo and in vitro, somatostatin-14 and somatostatin-immunoreactive substance (SSI) induce a juvenile status of the gland in adults and an activation of juvenile ones. These results suggest that they could regulate the growth and the repair of the shell and that the mantle edge gland might be considered as one of their tissue targets.

Molecular and biochemical study of the presence and synthesis sites of methionine enkephalin-like substances in the circumoesophageal ganglia, the ovotestis and the tentacles of the snail.

In situ hybridization with an oligonucleotidic 35S-labelled probe complementary to the 388-435 coding region of rat proenkephalin mRNA seems to show a colocalization between synthesis and storage sites of methionine-enkephalin(MK)-like substances only in young cellular stages of the ovotestis and in several neurons of cerebral, parietal and pleural ganglia. No positive signal can be detected neither in pedal and visceral ganglia nor in the tentacular collar cells, in spite of previous immunocytochemical data. Radioimmunoassays carried out on acidic extracts of the same organs confirm the molecular results and lead us to conclude to the presence of substances strongly related to MK in the ovotestis as well as in the circumoesophageal ganglia (COG), and to ascertain that the MK-positive tentacular collar cells do not contain authentic MK.

Immunocytochemical localization of insulin-related peptide(s) in the central nervous system of the snail Helix aspersa Müller: involvement in growth control.

1. The presence of insulin-like substances has been demonstrated by immunocytochemistry in the central nervous system of the snail Helix aspersa. 2. The immunopositivity has been observed especially in the large perikarya of the mesocerebral green cells [the cerebral green cells (CeGC) stained in green by the alcian blue:alcian yellow technique]. 3. The removal of either the mesocerebrum or the CeGC stops the growth of the snail and induces the increase of the glycogen content in the mantle edge. 4. Our results show the existence of insulin-like material in the neurosecretory cells. Previous data having demonstrated the presence of specific binding sites to insulin in the cephalic ganglia of Helix aspersa, one may suggest that insulin could play a neuromodulatory or a neurotransmittory role in the central nervous system and might control the growth.

[Demonstration of the presence and localization of the synthesis of a somatostatin-like substance by immunocytochemistry and hybridization in situ in the brain of Helix aspersa]. / Mise en évidence de la presence et des lieux de synthèse d'une substance apparentée à la somatostatine par immunocytochimie et hybridation in situ dans le cerveau d'Helix aspersa.

The immunocytological method has revealed the presence of somatostatin-like substance (SSI) in the brain of the snail Helix aspersa Müller. The Cerebral Green Cells (CeGC) in the mesocerebron and some neurons in parietal and visceral ganglia react positively with an antibody raised against Vertebrate somatostatin-14. The hybridization in situ with an oligonucleotide probe labelled with 35S-dATP complementary to the 3'-coding region of rat preprosomatostatin mRNA seems to show a colocalization between synthesis and stocking sites of SSI in the nervous ganglia. These results suggest for the first time that the codage of a SSI seems to be realized in the same way in Helix aspersa and Mammals.

Presence of cells and fibers immunoreactive toward antibodies to different peptides or amine in the digestive tract of the snailHelix aspersa.

An immunocytological study of four different parts of the gut of Helix aspersa clearly demonstrates the presence of many cells and fibers immunoreactive toward antibodies directed to vertebrate (α, ß-endorphin, α, ß-MSH, ACTH 1-24 and ACTH 17-39, met-enkephalin, somatostatin, insulin, glucagon, P.P., serotonin) or invertebrate (FMRF-amide) peptides. These results are evidence of the presence of different substances related to known peptides or amines in the epithelial and connective tissue cells and nerve fibers of the snail gut. Immunocytochemistry may help to elucidate the morpho-functional characteristics of the enteroendocrine cells of H. aspersa.

[Method of isolating FMRF-amide-like substances in the snail, using affinity chromatography coupled with HPLC]. / Mise au point d'une méthode d'isolement des substances FMRF-amide-like de l'Escargot, utilisant la chromatographie d'affinité couplée à la CLHP.

An extraction procedure of FMRFa-like substances from brain of Helix aspersa was developed. It consists of using affinity chromatography coupled with reverse phase HPLC. Three synthetic peptides (FMRFa, pQDPFLRFa, Met-enkephalin) were used to evaluate the specificity and yield of the affinity column. Its efficiency was tested by use of snail brain extracts. The results showed that this method is efficient and reproducible.

Variations of somatostatin-like immunoreactivity in the circumoesophageal ganglia, the hepatopancreas, the mantle edge, and the hemolymph of shell-repairing snails (Helix aspersa).

Immunocytology and radioimmunoassays demonstrate the presence of immunoreactive somatostatin-like (IR-SOM-LI) material in the tissues of young or adult snails Helix aspersa bred under short or long day in controlled artificial conditions. Neurosecretory cells in the circumoesophageal ganglia and in the digestive gland, small fibers in the hepatopancreas, and small granules in the mantle epithelial cells are immunoreactive toward antisomatostatin. In all experimented animals shell fractures induce variations of the IR-SOM-LI concentrations in all assayed tissues whatever the lighting conditions were underwent. These findings support the hypothesis that in Helix one or more substances related to the vertebrate tetradecapeptide is (are) involved in the repair processes but that the storage and the metabolism may be different during the biological cycle of the snails. These results are compared with those previously published using different gastropods and different methods.

Evidence for peptidergic innervation of the endocrine optic gland in Sepia by neurons showing FMRFamide-like immunoreactivity.

The innervation of the endocrine optic gland of Sepia, which controls sexual maturation, was studied by immunocytochemistry. Anti-FMRFamide (Phe-Met-Arg-Phe-NH2) serum revealed immunoreactive neurons in the olfactory and basal-dorsal lobe of the supra-esophageal brain mass. The axons of these neurons form a network from which fibers run to the optic gland. The fibers form many varicosities on the glandular cells, indicating synaptic innervation. Apparently, the two brain lobes containing the immunopositive cells function as a unit where visual and olfactory cues are integrated to regulate the endocrine activity of the optic gland.

Immunocytochemical and ultrastructural evidence for supra- and subesophageal localization of the dorsal-body cells of the snail Helix aspersa.

Dorsal-body endocrine cells (DBEC) of the snail were studied by means of immunocytochemical and electron microscopic methods at different times of the reproductive cycle. They specifically bind the anti-methionine-enkephalin (vertebrate--opioid-pentapeptide) antibody and are located not only near the cerebral ganglia but also in the connective tissue surrounding the subesophageal ganglia. Ultrastructural characteristics of these subesophageal cells, however, confirm their clear identity with the previously described supraesophageal cells. The quantitative variations of their immunoreactive content allow us to postulate a likely involvement in reproductive physiology (mating and egg laying). These observations prove that the distribution of the classical "dorsal-body cells" is more extensive than has been admitted until now and that they synthesize methionine-enkephalin-like substance(s).

[Immunocytochemical detection of substances related to methionine enkephalin in the tentacles and food of the snail (Helix aspersa)]. / Détection par immunocytochimie de substances apparentées à la méthionine-enképhaline dans les tentacules et dans le pied de l'escargot petit-gris (Helix aspersa).

Immunocytochemistry reveals the presence of methionine-enkephalin-like substance(s) in the collar cells of the two kinds of tentacles and in the foot of the snail Helix aspersa. The density of the immunoreactive material is higher in young animals than in adults. The greater part of the substance(s) is released at the surface of the epidermis and probably mixed with the mucus. A possible neuroendocrine and/or neuromodulatory function can be considered especially for the collar cells connected with the tentacular ganglia.

Immunocytological localization of a somatostatin-like substance in the brain of the giant slug, Limax maximus L.

Immunocytological tests reveal the presence of a somatostatin-like substance in perikarya and axons in the brain of the giant slug Limax maximus L. Controls carried out on adjacent sections with absorbed antiserum or different antibodies raised against several biologically active peptides of vertebrates (ACTH-17-39, alpha- and beta endorphin, alpha- and beta MSH, methionin-enkephalin, TRH) demonstrate the specificity of the "staining". However, some cells are both somatostatin- and FMRF-amide-positive. In the cerebral ganglia, the right Z-area cells, responsible for the synthesis of the maturation hormone (MH) are strongly somatostatin-positive. These results suggest a similarity between the MH and the somatostatin-like material contained in the Z-area cells. The simultaneous presence of two peptides in one and the same cell, the nature (elementary granules or soluble product) of the material, and its site of release are discussed.

[Immunocytologic detection of material related to various vertebrate peptides in the nerve ring of the snail (Helix aspersa Müller)]. / Détection immunocytologique de matériel apparenté à différents peptides de vertébrés dans le collier nerveux de l'escargot (Helix aspersa Müller).

The immunocytological method applied to the brain of the snail (Helix aspersa Müller) reveals positive antigenic sites with several antibodies raised against vertebrate peptides: somatostatin, ACTH-17-39, beta-MSH, beta-endorphin, methionine-enkephalin. The distribution of the positive structures (perikarya, fibers) in the ganglia, in the commissure or in the cerebral vessels is given and the presence of these substances is discussed.

Immunofluorescent localization and ultrastructural characterization of gonadotrophe cells in the adenohypophysis of the barbary drake (Cairina moschata L.) using anti-chicken LH serum.

An indirect immunofluorescence technique and an anti-chicken LH serum were used to localize cells in the adenohypophyses of drakes at different stages of their breeding cycle, after castration, and after castration combined with thyroxine treatment. Immunofluorescent cells were distributed throughout both lobes of the adenohypophyses from control and experimental birds and were shown to be alcian blue positive. PAS negative, basophiles. Immunofluorescent cells were as numerous in castrated birds as in castrated birds treated with thyroxine. Adjacent thin and semi-thin sections were used to study the cells binding anti-LH serum at light microscope and ultrastructural levels. The cells contained spherical granules with variable densities and diameters ranging between 40 and 280 nm in the rostral (=cephalic) lobe, and between 60 and 260 nm in the caudal lobe. The light microscope and ultrastructural observations showed that the anti-LH serum binds to cells which have been classified by other authors in the Pekin duck, quail and pigeon as TSH producing delta cells. The experimental technique used did not permit a distinction to be made between cells producing FSH and LH.

[Autoradiography and tritiated thymidine labeling during spermatogenesis of the Barbary Duck (Cairina moschata L.)]. / Etude par autoradiographie et marquage à la thymidine tritiée de la durée de la spermatogenèse du Canard de Barbaire (Cairina moschata L.).

Using autoradiographic method (intraveinous of 1 mCi-3H thymidine/kg of body weight) the authors show that meiotic prophase of the spermatogenesis of the Barbary drake (Cairina moschata L.) lasts 5 days 1/4; the first labeled gametes appear in the ejaculate at the end of the 12th day.

[Autoradiography after tritiated thymidine labeling of the abortive gametogenesis in hybrid male ducks from the cross male Pekin X female Barbary]. / Etude autoradiographique après marquage à la thymidine tritiée de la gamétogenèse abortive du Canard mâle hybride du croisement mâle Pékin X femelle Barbarie.

This autoradiographic data concludes that meiotic prophase of the abortive gametogenesis of the sterile hybride drake (from the mating Peking male = Anas platyrhynchos L. X Barbary female = Cairina moschata L.) lasts 5 days 1/4. 6 days after the injection of the marker the seminiferous tubules contain numerous labeles necrosing cells but never typical elongated spermatids.

[Abortive spermatogenesis in the hybrid duck (cross of male Anas platyrhynchos and female Cairina moschata). Ultrastructural study]. / Spermatogenèse abortive chez le canard hybride (du croisement Anas platyrhynchos mâle x Cairina moschata femelle). Etude ultrastructurale.

The ultrastructural study of the abortive spermatogenesis of the hybrid drake (cross: male Anas platyrhynchos L. X female Cairina moschata L.) shows the following characteristics: 1) deep anomalies during the pachytene stage (meiotic prophase) where the chromosomes form more or less irregular packs close to the nuclear enveloppe; 2) the total lack of 2nd spermatocytes and in their place were found: --either some cells with lobed or fragmented nuclei in a cytoplasm filled with numerous annulate lamella, lipid droplets and centrioles, --or necrosing cells, with vacuolised chromatin close to the inner side of the nucleus and with abundant smooth endoplasmic reticulum vesicles and dark mitochondria; 3) numerous Sertoli cells overloaded with lysosomes. The mitochondria of the spermatogonia, the basal membrane of seminiferous tubules and the Leydig cells have the same structure that exists in the fertile Barbary drake testis.