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PubMed1975

Trypsin-kallikrein isoinhibitor K (type Kunitz) from snails (Helix pomatia). Purification and characterization.

A basic proteinase inhibitor, isoinhibitor K, was purified by SE-Sephadex C-25 column chromatography from the mixture of acid-stable and heat-stable isoinhibitors of the snail (Helix pomatia). Isoinhibitor K is homogeneous in polyacrylamide gel, cellulose acetate and polyacrylamide-dodecylsulfate electrophoresis. From the electrophoretic mobility in dodecylsulfate-polyacrylamide gel and apparent molecular weight of 6500 +/- 200 was estimated. From the amino acid composition the inhibitor consists of 58 amino acid residues. It contains three disulfide bridges, a C-terminal valine and a lysine residue at the reactive site. Isoinhibitor K inhibits the enzymes: bovine trypsin and chymotrypsin, porcine plasmin and pancreatic kallikrein, the trypsin-like component of Streptomyces griseus proteinase-pronase E, and fungi proteinase K from Tritirachium album Limber, which is only inhibited very slightly in contrast to the effect of the mixture of isoinhibitors. The inhibitory effect of isoinhibitor K against these enzymes is compared to that of the mixture or of other isoinhibitors. The following enzymes are not inhibited by isoinhibitor K: Aspergillus proteinase P and alkaline bacillus proteinase 2231 (Röhm), which both are inhibited by the mixture of isoinhibitors. Porcine elastase, bacterial proteinase N (M) (Röhm), and a trypsin-like proteinase from wheat are not inhibited, porcine acrosin and porcine serum kallikrein only to a very minor extent by the mixture of isoinhibitors. Reactive-site peptide-bond cleavage during inhibition could not be detected. Thus, the inhibitory behaviour is just as broad in specificity and as unusual as that of the trypsin-kallikrein inhibitor (Kunitz) from bovine organs. The N-terminus is blocked by pyroglutamic acid. Isoinhibitor K is the main component of the isoinhibitors secreted into the mucus and amounts to 35-40% of the mixture.

Amino Acids↗
PubMed1979

The effect of adenosine triphosphate, magnesium chloride and phospholipids on crystal formation in the demineralized shell-repair membrane of the snail, Helix pomatia L. An in vitro study.

The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl2) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO3) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0 mM ATP. The inhibitory effect of Mg2+ on CaCO3 crystal formation was diminished by reduction of the concentration of MgCl2 in the incubation solution. Thus, after incubation for only 24h, 1.0 mM MgCl2 promoted the formation of birefringent CaCO3 crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl2 and phospholipids in the recalcification of shell-repair membrane is discussed.

Adenosine Triphosphate↗
PubMedDate not supplied

Electron cytochemical study of carbohydrate components in different types of cultured glial cells of snail Helix pomatia.

Using a variety of colloidal gold-labelled lectins with different sugar specificities, the structure and topography of carbohydrate determinants of the surface membrane of in vitro cultured glial and nerve cells of the snail Helix pomatia have been electron cytochemically studied. Heterogeneity of carbohydrate pools among different types of glial cells and between glial and nerve cells was established. It was found that satellite glial cells having the ultrastructural signs of cells with high metabolic level (type II cells) selectively bind GNA which is specific to terminal alpha-D-mannose residues and do not bind other mannose-specific lectins, Con A and LCA. GNA determinants are absent in satellite type I glial cells, fibrous glial cells, microglia and neurons. It has been found that glial cells (satellite type I and II glial cells, filamentous glial cells and microglial cells) do not bind PVA and LABA. LTA did not bind to any glial cells and binds weakly to neurons. Con A and WGA determinants which are abundant on the neurons are completely absent on satellite type II glial cells but present on satellite type I glial cells and filamentous glial cells. Microglial cells contain Con A and LCA determinants and the density of PNA determinants on these cells is the highest compared to other types of glial cells or neurons. It is concluded that some lectin determinants (for RCA-1, PNA, LPA) are present on all types of glial cells, while another determinant (GNA) is specific for a definite type of glial cells and can serve as a marker of these cells. The role of specific carbohydrate determinants in the functioning of a neuron-glial complex is discussed.

Animals↗
PubMed1989

Evidence for a light compensation of the inhibition of reproduction by low temperatures in the snail Helix aspersa. Ovotestis and albumen gland responsiveness to different conditions of photoperiods and temperatures.

Four combinations of photoperiods and temperatures have been tested on the reproduction and on the activity of the genital apparatus of the snail Helix aspersa. The results show some interaction between photoperiod and temperature and reveal a predominant effect of photoperiod that compensates for the negative effect of low temperatures. A combination of long day (LD, 18L:6D) and a temperature of 20 degrees C is the most favorable condition for egg-laying as well as for the effective functioning of the ovotestis and albumen gland. In contrast, during a short day (SD, 8L:16D), egg-laying is completely inhibited at 15 degrees C and partly inhibited at 20 degrees C; the differentiation of gametes can be observed in the ovotestis, but the mature oocytes are not released and thus they degenerate. In the albumen gland, an important synthetic activity is noticed, but cellular multiplication and the release of the secretory products decrease. These results suggest that short photoperiods induce inhibition of the (neuro)endocrine centers responsible for the control of both ovulation and egg-laying.

Animals↗
PubMed1985

[Control of secretory activity of multifid glands by castration and grafting in the snail Helix aspersa].

Castration of juvenile snails results in a cessation of multified gland cell differentiation. Many months later, the cells still resemble polarized epithelial cells and show no signs of secretory activity. Transplantation of nondifferentiated cells from multifid gland of castrated snails into normal adults initiated secretory activity. The experiments show that the presence of the gonad is essential for the multifid glands to develop secretory activity and that its influence is mediated by an endocrine factor which is transported in the hemolymph.

Animals↗
PubMed1977

Cytochemical localization of acid phosphatase in regenerated and dark-adapted eyes of a snail, Helix aspersa.

The role fo the Golgi apparatus and the Golgi-endoplasmic reticulum-lysosomal complex (GERL) in the formation of lysosomes in the photosensory cells of regenerating and dark-adapted eyes of the garden snail Helix aspersa was examined with use of acid phosphatase as a marker enzyme. In newly regenerated eyes, lead reaction deposit was restricted to the cisternae of GERL, a few small vesicles and some large secondary lysosomes. Dark-adapted sensory cells, on the other hand, were characterized by a heavy reaction product in the hypertrophied Golgi cisternae and GERL. Primary lysosomes were packaged by GERL cisternae in both the regenerating and the degenerating dark-adapted eyes. In the latter, these lysosomes may have been produced also by the Golgi apparatus. No reaction product was found in the rough endoplasmic reticulum, microvilli, or large aggregations of 800 A photic vesicles in either kind of eye. In this study the lytic activity in differentiating sensory cells was significantly lower than that in degenerating cells suggesting that the increase in lysosomal activity in the latter was due to the absence of light. The effects of long dark-adaption appear to be: 1. decrease in the production of photic vesicles; 2. increase in the formation of lysosomes; and 3. breakdown of photic vesicles by lysosomal activity.

Acid Phosphatase↗
PubMed2004

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria.

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).

Acclimatization↗
PubMed1992

The primary structure of neuropeptide F (NPF) from the garden snail, Helix aspersa.

Neuropeptide F (NPF), originally isolated from the sheep tapeworm, Moniezia expansa, consists of 39 amino acid residues terminating in a phenylalaninamide. An analogous neuropeptide has been isolated and sequenced from extracts of circumoesophageal ganglia of the garden snail, Helix aspersa. This neuropeptide exhibits partial primary structural similarity to members of the vertebrate neuropeptide Y (NPY)/pancreatic polypeptide (PP) superfamily. NPF is thus of widespread occurrence in the nervous systems of invertebrates from different phyla and may represent the phylogenetic precursor of the vertebrate NPY/PP superfamily.

Amino Acid Sequence↗
PubMed2001

Mannan-degrading enzymes purified from the crop of the brown garden snail Helix aspersa Müller (Gastropoda Pulmonata).

Two mannan-degrading enzymes were purified from the crop of the terrestrial snail Helix aspersa Müller. The crude extracts were taken from dormant (for 4 months) snails. The enzymes were a betaD-mannanase of 37.4 +/- 0.3 kDa (EC 3.2.1.78) and a betaD-mannosidase of 77.8 +/- 1.9 kDa (EC 3.2.1.25). Both enzymes degraded insoluble mannan, releasing either mannose only (beta-mannosidase) or oligosaccharides, possibly mannotriose and mannopentaose (beta-mannanase). The beta-mannanase had a typical endo-activity pattern, while the beta-mannosidase was an exoenzyme. The incubation of both enzymes with mannan increased the catalysis by 83%. The best synergy was found with 75% mannosidase combined with 25% mannanase. The beta-mannanase also hydrolysed beta-linked heteromannans and its affinity for different galactomannans was studied. The Km values, varying from 2.89 +/- 0.47 mg x ml(-1) to 0.26 +/- 0.01 mg x ml(-1), revealed the inhibitory effect of the alphaD-galactosyl residues released. The beta-mannosidase was acidic (optimum pH = 3.3) and heat-sensitive (50% residual activity at 42 degrees C after 5 min of pre-incubation), while the beta-mannanase remained stable until 48.5 degrees C (50% residual activity) and over a pH range of 4.3-7.5. The properties of these mannanolytic enzymes are discussed in this paper compared with those purified in other gastropods and in a bacterium, Enterococcus casseliflavus, a quite similar strain previously isolated from this snail intestine. The occurrence of thermostable enzymes in H. aspersa digestive tract could be a zootechnic parameter of great importance for snail farming. J. Exp. Zool. 290:125-135, 2001.

Animals↗
PubMed1993

Identification and quantification of gamma-glutamyl conjugates of biogenic amines in the nervous system of the snail, Helix aspersa, by gas chromatography-negative-ion chemical ionisation mass spectrometry.

The gamma-glutamyl conjugates of p-octopamine and dopamine were identified unambiguously for the first time and quantified in a single cerebral ganglion or pleural plus pedal ganglia of the snail, Helix aspersa, by gas chromatography-negative-ion chemical ionisation mass spectrometry. A new method was used for synthesis of gamma-glutamylamine standards. The concentration of gamma-glutamyltyramine was found to be low in the tissues, therefore it was used as an internal standard. The gamma-glutamylamines were extracted with water and derivatised with pentafluoropropionic anhydride and trifluoroethanol. Under negative-ion chemical ionisation conditions, the trifluoroethyl and pentafluoropropionyl derivatives produced significant ions which were sufficiently abundant to be suitable for selective ion monitoring. The method had a limit of detection of ca. 80 pg of gamma-glutamyl conjugate per tissue and calibration curves were linear over the range examined.

Animals↗
PubMed1976

Properties of a facilitating calcium current in pace-maker neurones of the snail, Helix pomatia.

1. Simultaneous measurements of local voltage clamp currents from patches of soma membrane and K activity at the soma surface were used to analyse the time and voltage dependence of the slow inward current in bursting pace-maker neurones of the snail (Helix pomatia). 2. At low levels of depolarization (less than or equal to mV) a net inward current is recorded simultaneously with an efflux of K ions from the cell. 3. With larger depolarizations (20-170 mV from holding potential of -50 mV) the deficit in net outward charge transfer compared with K efflux and the appearance of inward-going tail currents following repolarization, reveal a persistent inward-going current also under these conditions. This inward current is carried primarily by Ca ions, as demonstrated by its voltage dependence (a minimum at about + 115 mV) and its disappearance in Co-Ringer. It is identified with the slow inward Ca current Iin slow (Eckert & Lux, 1976). 4. The inward current predicted from comparisons of current trajectories reaches a maximum at 15-20 msec (for depolarizations from -50 to 0 mV) and gradually declines with sustained depolarization. 5. Partial inactivation is removed by repolarization to -50 mV and the Ca dependent deficit is greater in the sum of repeated voltage clamp pulses than during sustained depolarization. It is largest for pulses of 25-100 msec duration, decreasing as pulse duration increases. 6. Responses to repeated activation with 100 msec pulses with different repolarization intervals reveal a minimum Iin slow at short intervals (e.g. 20 msec) due to failure to remove partial inactivation. At intermediate intervals (e.g. 200-400 msec) Iin slow shows facilitation. This is revealed in calculations of the net charge transfer and current deficits and is also shown in the tail currents following repolarization. The deficit increases progressively with repetitive stimulation. With longer intervals (e.g. 800-1000 msec) defacilitation during repeated stimulation after the first two pulses is revealed in calculations of deficits, current trajectories and in the tail currents. 7. Although facilitation depends on duration of repolarization between pulses, increasing intermediate hyperpolarizations from the holding potential of -50 mV are usually ineffective in increasing Iin slow. Strong preceding hyperpolarization can even decrease the magnitude of Iin slow and prevent its facilitation with repetitive stimulation,whereas preceding depolarizing pulses can increase Iin slow without preventing its facilitation with repetitive stimulation. 8. The properties of Iin slow are contrasted with previously described membrane conductances and compared with properties attributed to Ca fluxes in other systems.

Action Potentials↗
PubMed1984

Intracellular protein kinase and calcium inward currents in perfused neurones of the snail Helix pomatia.

Changes in the amplitude of the calcium inward current caused by intracellular administration of tolbutamide (an inhibitor of the cyclic AMP-dependent protein kinase activity) or catalytic subunits of cAMP-dependent protein kinases from rabbit myocardium were studied on internally perfused nerve cells of the snail, Helix pomatia. Intracellular administration of 7 mM tolbutamide caused a rapid decline of the amplitude of the calcium current that had been stabilized by theophylline; the effect was practically completely reversible. In contrast, addition to the perfusing solution of exogenous catalytic subunits of cyclic AMP-dependent protein kinases (about 0.7 microM of protein) together with 2 mM adenosine 5'-triphosphate and 3 mM MgCl2, led to stabilization of the calcium conductance of the cell membrane or restored it if it had declined during the perfusion with basic solution. The effect depended largely on the presence of adenosine 5'-triphosphate. Its time course was very slow (dozens of minutes) due probably to slow diffusion of the protein inside the cell. Heat-inactivated catalytic subunits did not produce such a stabilizing or restoring action on the calcium conductance. The results substantiate the suggestion that the normal functioning of calcium channels depends on phosphorylation catalyzed by cyclic AMP-dependent protein kinases.

Adenosine Triphosphate↗
PubMed1995

Conversion of adenosine(5') oligophospho(5') adenosines into inosine(5') oligophospho(5') inosines by non-specific adenylate deaminase from the snail Helix pomatia.

Until now, the catabolism of adenosine(5')triphospho(5')adenosine (Ap3A) and adenosine(5')tetraphospho(5')adenosine (Ap4A) has been thought to commence with either hydrolytic or phosphorolytic cleavage of their oligophosphate chains, depending on the organism. Here, we show that in the extracts from the retractile 'foot' of the snail Helix pomatia deamination predominates; the adenosine moieties of these and other adenosine(5')oligophospho(5')adenosines (ApnAs) undergo successive deamination leading, via an inosine(5')oligophospho(5')adenosine (IpnA), to the corresponding inosine(5')oligophospho(5')inosine (IpnI). The reactions are catalyzed by the non-specific adenylate deaminase described earlier (Stankiewicz, A.J. (1983) Biochem. J. 215, 39-44). We describe TLC and HPLC systems which allow the separation of any of the deaminated derivatives from its parent compound; Ap2A, Ap3A, Ap4A or Ap5A. The Km values for these substrates are 20, 22, 32 and 39 microM, respectively, whereas the Km for 5'-AMP is 12 microM. Relative substrate specificities for these compounds amount to 25, 18, 14, 7 and 100. The enzyme was shown also to deaminate phosphonate and thiophosphate analogues of Ap3A.

AMP Deaminase↗
PubMed1993

Neurophysiological correlates of tactile stimulus-induced whole-body eversion, a novel type of behavior in the snail Helix pomatia L.

Repeated weak tactile stimuli to the exposed skin of the foot of withdrawn snails (Helix pomatia L.) result in a rapid whole-body eversion response not previously described. We studied the neurophysiological correlates of this novel type of behavior in semi-intact preparations consisting of the foot, pneumostome and mantle collar attached to the CNS. The pneumostome opening component of the eversion response is retained in semi-intact preparations and can be triggered by a series of weak tactile stimuli to the foot. The same stimuli also strongly excite a giant neuron (LPd7) in the pedal ganglion. This cell has axon branches in the anal and left pallial nerve which innervate the pneumostome and mantle collar. Intracellular stimulation of LPd7 causes the pneumostome to open. Bursts of spikes in LPd7 also lead to contractions of a specific group of muscles in the mantle collar innervated by the left pallial nerve. Both responses are retained when the CNS is bathed in high Mg2+/0 Ca2+ saline but the mantle response has a much shorter latency than the opening of the pneumostome. We conclude that LPd7 is an efferent neuron which causes opening of the pneumostome via a peripheral pathway in the mantle collar and may also be a motoneuron of muscles in the mantle collar. Besides its pneumostome-opener and putative mantle motoneuron function, the LPd7 cell also triggers movements of the foot which are also part of the eversion sequence in intact snails. The LPd7 cell receives excitatory input from neurons which trigger withdrawal reactions in active snails. The excitation is due to peripheral interactions between the two cell types and leads to opening of the closed pneumostome following brief aversive stimuli. We suggest that LPd7 is part of a novel putative neuronal network underlying the described eversive reaction to tactile stimuli.

Animals↗
PubMed1998

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.

Animals↗
PubMed1994

Central chemoreceptor stimulus in the terrestrial, pulmonate snail, Helix aspersa.

We studied the effect of hypercapnic and fixed acid central chemoreceptor stimulation on the pneumostome in the pulmonate snail, Helix aspersa. We found that focal stimulation of the central chemoreceptor area of the pulmonate snail brain with hypercapnic solutions more effectively increased the pneumostomal area than did fixed acid stimulation at the same extracellular pH. Disrupting intracellular pH regulation by inhibiting Cl- transport, either pharmacologically (DIDS) or by ion substitution (Cl(-)-free perfusate), enhanced pneumostomal responses to CO2. While maintaining a constant perfusate pH, addition of NH4Cl to the perfusate resulted in pneumostomal closure; whereas removal of NH4Cl from the bath resulted in pneumostomal opening. In conclusion, the ventilatory response to CO2 in H. aspersa does not require Cl- transport or conductance. Furthermore, changing pHi alone is an adequate stimulus for the central chemoreceptors in the snail.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Spatial analysis of allozyme and microsatellite DNA polymorphisms in the land snail Helix aspersa (Gastropoda: Helicidae).

The genetic structure of the land snail Helix aspersa was investigated for 21 populations collected along a road located in the polders of the Bay of Mont-Saint-Michel (Brittany, France), following a sampling scheme the area of which did not exceed 900 m in length. A total of 369 individuals were genotyped for five enzymatic markers and seven microsatellite loci. We used sequential hierarchical F-statistics at different spatial scales and spatial autocorrelation statistics to explore recent historical patterns involved in the observed genetic distribution. Whatever the statistics used, congruent levels of spatial genetic substructuring across loci were demonstrated, excepted for one allozyme locus. Overall spatial genetic arrangement matched in a substantial fashion theoretical predictions based on the limited dispersal power of land snails. Positive autocorrelation over short-distance classes may result from the development of genetically distinct patches of individuals organized in family-structured colonies. Therefore, spatial signatures of average I correlograms can be viewed as the expression of a stepping-stone model of population structure, sometimes involving external migrational events. Overall, the revealed pattern of population subdivision on a microgeographical scale was suggestive of a neighbourhood structure. Finally, microsatellite loci are especially suitable for the detection of small genetic clustering, and combining different classes of markers offers the potential to gain further insight into the description of spatial genetic variability over short temporal and geographical scales.

Animals↗