[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.

DARPP-32 like protein in specific snail (Helix aspersa) neurones.

Monoclonal antibodies to DARPP-32 recognise an antigen which is present in specific neurones in the snail (Helix aspersa). Consecutive sections 10-microns-thick processed for the localisation of DARPP-32 and tyrosine-hydroxylase immunoreactivity did not show a coexistence in any neuronal structures. DARPP-32 positive cells were, however, often morphologically closely associated with tyrosine-hydroxylase positive cells, implying a functional relationship consistent with the proposed role of DARPP-32. Immunochemical analysis of the DARPP-32 immunoreactive material in the snail nervous system shows that the substance has a molecular weight of 28 kDa and therefore different from the DARPP-32 protein found in the rat brain.

Distribution and characterization of pedal peptide immunoreactivity in Aplysia.

Pedal peptide (Pep) is a very abundant neuropeptide in Aplysia. A radioimmunoassay (RIA) was developed to quantify Pep-like immunoreactivity (IR-Pep) in tissue extracts. IR-Pep was present in very high concentrations in the central nervous system (CNS) and two peripheral tissues: the large hermaphroditic duct (LHD) and the foot. RIA of fractions from high-pressure liquid chromatography (HPLC) indicated that Pep itself was the predominant immunoreactive species in each of these tissues. Lower concentrations of Pep were found in a number of other peripheral tissues. Incorporation of labelled amino acid indicated that Pep was synthesized in the LHD, whereas Pep in the foot was synthesized primarily in central neurons and transported to the foot. IR-Pep was further localized by immunocytology. All peripheral IR-Pep appeared to be associated with neuronal fibers, most commonly varicose axons. Immunoreactive innervation of the LHD and foot was particularly dense but positive staining was also observed in other tissues including tegument, gill, gut, and heart, IR-Pep innervation in all tissues including the LHD appeared to be localized predominantly in muscular portions of the tissue. Spontaneous contractions of isolated LHD were accelerated by the application of Pep. Pep appears to act as a transmitter or neuromodulator at a number of different sites in Aplysia.

Structural studies on the galactan from the snail Helix pomatia.

1. The galactan of the snail Helix pomatia was subjected to two cycles of Smith-degradation and the resulting products were isolated by gel filtration and thin layer chromatography. 2. The structures of the low molecular weight oligosaccharides were elucidated being identical to those obtained from Lymnaea stagnalis galactan. However, the quantities released differed significantly between the two species. The high molecular fractions comprising about 66% of the material were not obtained in a similar degradation of the Lymnaea stagnalis galactan. 4. Thus the observed structural differences can explain easily the species-specific reactivity among the two polysaccharides seen earlier with lectins, enzymes and antibodies.

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.

[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.

[Distribution of the LPa3 and PPa3 neuronal processes in the pedal ganglia of the edible snail]. / Raspredelenie otrostkov neironov LPa3 i PPa3 v nervakh pedal'nykh gangliev vinogradnoi ulitki.

By means of intracellular injection of the dye Lucifer yellow processes were revealed of the snail command neurones of escape behaviour LPa3 and RPa3 in pedal nerves ipsilateral to these neurones. A process of the neurone LPa3 was also found in the contralateral Nervus cutaneus pedalis secundus. Blockade of central chemical synapses by cadmium chloride did not lead to disappearance of motor reactions evoked by electrical stimulation of LPa3 and RPa3 neurones both on ipsi- and contralateral sides of the animal foot; this allowed to suggest a presence in the contralateral muscular pedal nerves of these neurones processes unrevealed by the used dyeing method.

[Peripheral distribution of a neuropeptide recognized by an antiserum raised against the mammalian CRF41, in the mollusc Helix pomatia]. / Distribution périphérique d'un neuropeptide reconnu par un anti-sérum dirigé contre le CRF41 des mammifères, chez le mollusque Helix pomatia.

Immunocytochemical methods using an antiserum raised against ovine corticoliberin revealed perikarya and processes in the central and peripheral nervous system of the Pulmonate Gastropod Helix pomatia. The coexistence of immunoreactive nerve fibres and primary sensory neurons in the intestinal wall and in the tentacles appeared particularly significant. The distribution of this peptide suggests that it could act as a sensory neurotransmitter at the central and peripheral levels.

FMRFamide receptors in Helix aspersa.

An in vitro receptor binding assay and an isolated heart bioassay were developed and used to characterize the structure-activity relations (SAR) of FMRFamide receptors in a land snail, Helix aspersa. In the radioreceptor assay, binding of 125I-desaminoTyr-PhenorLeu-Arg-Phe-amide (125I-daYFnLRFamide) at 0 degrees C to Helix brain membranes was reversible, saturable, and specific, with a KD of 14 nM and a Bmax of 85 fmol/mg brain. A lower affinity site was also observed (KD = 245 nM; Bmax = 575 fmol/mg brain). In the heart bioassay, daYFnLRFamide and other FMRFamide analogs increased myocardial contraction force. The SAR of cardiostimulation correlated with the specificity of high affinity 125I-daYFnLRFamide binding to brain and heart receptors. The SAR was also similar to that described for other molluscan FMRFamide bioassays, except for a marked preference for N-blocked analogs. Peptides with N-terminal extensions of desaminoTyr, Tyr, Tyr-Gly-Gly, and acetyl, exhibited the highest potency in both radioligand displacement and cardiostimulation. The endogenous Helix heptapeptide analogs of FLRFamide (pQDP-, NDP, and SDP-FLRFamide) were stimulatory on the heart at low doses, but were inhibitory at moderate to high doses. These peptides were 20 times weaker than FMRFamide in both the brain and heart receptor binding assays, with IC50s about 10 microM. The results suggest that the effects of FMRFamide in Helix are receptor-mediated, and that the heptapeptides do not interact at FMRFamide receptors.

Localization of FMRFamide-like peptides in the snail Helix aspersa.

The distribution of FMRFamide-like material in the gastropod mollusc, Helix aspersa, was studied by radioimmunoassay (RIA) and immunocytochemistry. Most of the RIA activity was concentrated in the central nervous system, the male reproductive tract, the tentacles and the posterior digestive system (Table 1). The density of FMRFamidergic perikarya, nerves and nerve varicosities in the muscle tissue of all these regions, as indicated immunocytochemically (Fig. 2), was well correlated with the distribution as determined by RIA. Gel chromatography of each extract resolved two peaks of FMRFamide-like immunoreactivity (Fig. 3). The first of these was further analysed by high-pressure liquid chromatography (HPLC), and the components included two major immunoreactive peaks identifiable, both by their retention times and their effects on the radula protractor muscle of Busycon contrarium, as the known peptides FMRFamide and pQDPFLRFamide (Figs 4-6). The second peak from gel chromatography gave only a single peak, distinct from that of FMRFamide and pQDPFLRFamide, in two HPLC systems (Fig. 7), but it did not behave like a competitive ligand in the FMRFamide RIA. Moreover, its immunoreactivity, unlike any peptides we tested, was not affected by carboxypeptidase Y (Fig. 8), and it was not active on the radula protractor muscle. Thus, it is certainly not an FMRFamide-like peptide. We conclude that Helix aspersa contains at least two FMRFamide-like peptides, FMRFamide and pQDPFLRFamide. These peptides appear to act both as neurohormones and as neurotransmitters or modulators in the central ganglia, reproductive, digestive, muscular and circulatory systems.

New method for calculating pHi from accurately measured changes in pHi induced by a weak acid and base.

From the combined Henderson-Hasselbalch relations for a weak acid and a weak base, we derive an expression for steady-state intracellular pH(pHi). The derivation requires only accurate measurements of the pHi changes when the two electrolytes are applied, rather than absolute values. The method is based on the assumption of equilibrium of the neutral forms of the two compounds across the plasma membrane, that is, absence of permeation of the charged forms. It is also assumed that no significant pHi regulation takes place, that there is no significant permeability to intracellular buffers and that intracellular buffering power, over the measured span of pHi, is the same. This precludes the use of CO2/bicarbonate buffered systems. We find that under our experimental conditions steady-state pHi values calculated in this way agree closely with those measured directly with pH-sensitive microelectrodes both in snail neurones and crab muscle. The method would allow the intracellular calibration of other pHi measuring techniques.

[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.

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa.

A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles. Within the central ganglia many neurons, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata. Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.

Biochemical and immunological evidence for a cardiodilatin-like substance in the snail neurocardiac axis.

Cardiac hormones, which have been isolated recently from mammalian atria, are potent regulatory peptides of blood pressure and blood volume. By using biochemical and immunological methods to determine cardiac hormones of the cardiodilatin family, this type of peptide hormone was detected in a neurosecretory system projecting from the subesophageal ganglion to the heart of the snail. The cardiodilatin-like molecule was characterized by its biological effects on mammalian vascular smooth muscle, by radioimmunoassay combined with high-performance liquid chromatography, and by immunocytochemistry. In mammals cardiodilatin-like peptides appear to serve purely endocrine functions. In contrast, in the snail they are present in a neuroendocrine system, the function of which remains to be established.

Primary structure of the low-molecular-weight carbohydrate chains of Helix pomatia alpha-hemocyanin. Xylose as a constituent of N-linked oligosaccharides in an animal glycoprotein.

alpha-Hemocyanin of Helix pomatia is a copper-containing glycoprotein which serves as an oxygen carrier in the hemolymph. Its carbohydrate moiety has as constituents fucose, xylose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine, and N-acetyl-glucosamine residues. Alkaline borhydride did not split off any carbohydrate material, suggesting the absence of O-glycosidic chains. The N-glycosidic carbohydrate chains of this glycoprotein were liberated by hydrazinolysis of a Pronase digest then fractionated as alditols on Bio-Gel P-4. The fractions containing the low-molecular-weight glycans were investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar and methylation analysis. The largest, and most abundant, compound was established to be: (Formula: see text). Another compound was characterized as the afuco analogue of this structure. H. pomatia alpha-hemocyanin is the first example of an animal glycoprotein having xylose as a constituent of N-glycosidic carbohydrate chains.

[Electrophoretic detection of protein fractions in extracts of the ocular tentacles of Helix aspersa Müller, 1774 (Gasteropoda, Pulmonata, Helicidae)]. / Détection électrophorétique des fractions protéiques des extraits de tentacules oculaires d'Helix aspersa Mûller, 1774 (Gasteropoda, Pulmonata, Helicidae).

The extracts from ocular tentacles of Helix aspersa hare a heterochronic inhibitive action on the development of albumen gland. In view of elucidating the origin of this heterochrony, we make an electrophoretic study of the different extracts from juveniles, adults in activity, in natural sleep and awakning. This study has allowed us to spot 19 proteic fractions common to the four extracts of the tentacles under study, but these fractions show only differences in concentration. Particularly, the proteinic fractions 7 and 8 are more concentrated in the extract of juveniles snails tentacles than in the extract of adults snails. This difference in proteic concentration could explain the heterochronic inhibitive action of ocular tentacles extracts on the development of albumen gland of this snail.