Opsonizing effect of serum and albumin gland extracts on the elimination of human erythrocytes from the circulation of Helix pomatia

The removal of human erythrocytes of the A₁ and B types from the circulation of the gastropod Helix pomatia follows an exponential curve. The elimination of the nonself particles is apparently dependent on serum opsonins as preincubation of A₁ and B erythrocytes in Helix serum increases the rate of their clearance. This conclusion is supported by our finding that secondary doses of nonsensitized A₁ erythrocytes injected 12–19 hr after a similar primary dose are cleared very slowly; however, the clearance rate returns to normal if erythrocytes comprising the second dose are preincubated with Helix serum. Furthermore, the elimination of second-dose A₁ erythrocytes is strongly enhanced after their pretreatment with agglutinating extracts of the albumin glands from H. pomatia and Cepaea (Helix) nemoralis. On the other hand, no opsonizing effect was obtained by pre-incubating A₁ erythrocytes in the agglutinating extract of the sponge Aaptos papillata.     

Identification of agglutinin receptors on hemocytes of Helix pomatia

The attachment of opsonized foreign particles to phagocytic cells indicates the occurrence of opsonin receptors on the surfaces of the phagocytes. There is good evidence that naturally occurring hemagglutinins may serve as opsonins in invertebrates. To prove the occurrence of agglutinin receptors on the hemocytes of an invertebrate, the interaction of various agglutinins with Helix pomatia hemocytes was investigated. A positive agglutination reaction was obtained with Ricinus, Axinella, anti-H_eel, Ulex, concanavalin A, and Limulus agglutinins. The known specificities of these agglutinins and the influence of carbohydrases on the agglutinability of Helix cells have led to the conclusion that the carbohydrate components of the binding sites include galactose, fucose, mannose or glucose or both, and N-acetylneuraminic acid or polygalactose.     

Discriminative ability and function of the immunobiological recognition system of the snailHelix pomatia

Summary The internal defense mechanism ofHelix pomatia discriminates between different types of foreign cells as demonstrated by determinations of their clearance rates. The rate of elimination is not dependent on the size of foreign cells but on their molecular surface properties. Circulating hemocytes are not involved in the first phase of the clearance event, which is characterized by an accumulation of nonself cells in the digestive gland, kidney and foot muscle ofHelix. Light microscopic studies of these organs reveal nonself cells to be attached to the membrane of cells lining hemolymph sinuses. The attachment of certain types of foreign cells is apparently mediated by opsonins as their clearance depends on the opsonin level of the hemolymph, whereas others are cleared without involvement of opsonizing molecules. Membrane bound molecules of the latter type of nonself cells seem to directly interact with carbohydrate-specific combining sites on the membranes of cells of the sinus walls, as their binding can be inhibited by N-acetyl-galactosamine, and N-acetyl-glucosamine.The second phase of clearance apparently involves the attraction of circulating hemocytes by organtrapped foreign cells. The number of hemocytes in circulation decreases significantly, whereafter a rising percentage of hemocytes containing foreign cells can be observed in the circulation.     

Ovarian infection and fungal spore oviposition in the blackfly Prosimulium mixtum

In order to ascertain whether agglutinins can serve as links to bind hemocytes of Helix pomatia to mammalian erythrocytes, rosette-formation tests were performed. These involved pretreatment of H. pomatia hemocytes with each of 15 nonnative agglutinins and incubation of them with human erythrocytes. It has been found that, of the agglutinins tested, wheat germ agglutinin (WGA) as well as those from Ricinus communis, Axinella polypoides, Anguilla anguilla (anti-H_eet), concanavalin A, and Limulus polyphemus caused rosette formation with human erythrocytes. In addition, it has been found that a small number of H. pomatia hemocytes are capable of direct binding to erythrocytes of mice, rabbits, rats, and sheep.     

The reactivity of canine red cells with heterophile agglutinins having anti-A activity*

Heterophile A-reactive agglutinins, obtained from the seeds of Dolichos biflorus and from the snails Helix aspersa, Helix pomatia and Cepaea nemoralis, were tested for reactivity with the saliva and red cells of a random series of dogs. Saliva from dogs with the A-like Tr red cell antigen inhibited the agglutination of human A red cells by each of the reagents. However, none of the heterophile agglutinins distinguished Tr+ from Tr— red cells: the Dolichos agglutinin was non-reactive, while all of the snail reagents agglutinated both Tr+ and Tr— cells. It is concluded that the canine red cell bears a series of heterophile receptors which mask reactivity with the canine homologue of the red cell A-antigen, and that the restricted size of the binding sites of the agglutinins limits the informational significance of cross-reactivity obtained with these reagents.     

Two lectins on the surface of Helix pomatia haemocytes: a Ca2+-dependent,GalNac-specific lectin and a Ca2+-independent,mannose 6-phosphate-specific lectin which recognises activated homologous opsonins

Summary Haemocytes of the gastropod mollusc, Helix pomatia, possess on their surface a membrane-integrated GalNac-specific lectin which binds to and stimulates phagocytosis of GalNac-bearing target cells (human A erythrocytes) only in the presence of extracellular calcium ions. Target cells without GalNac moieties on their surface (human B and bovine erythrocytes) are not recognised. Helix haemocytes also possess a Ca²⁺-independent mannose-6-phosphate-specific lectin on their surface which, in the absence of extracellular calcium ions, enables recognition and phagocytosis of A rbc opsonised with agglutinins isolated from either the snail's albumin gland or serum. These opsonins, however, bind to host haemocytes only after binding to GalNac moieties on the surface of test particles. Our results indicate that such a ligand-specific opsonin/target cell interaction apparently induces a conformational change in the opsonin, resulting in exposure of mannose 6-phosphate moieties that are recognised by the Ca²⁺-independent lectin on the surface of the haemocytes.Abbreviations BSA bovine serum albumin - bv bovine - DAB 3-3-diamino-benzidine tetrahydrochloride - ELISA enzyme-linked immunosorbent assay - GalNac N-Acetyl-D-galactosamine - G6-P glucose 6-phosphate - HE haemocyte extract - HPA Helix pomatia albumin gland agglutinin - HPA PO peroxidase-labelled HPA - M6-P mannose 6-phosphate - ML monolayer - MLS monolayer supernatant - OPD orthophenylene diamine - PBS phosphate buffered saline - PMSF phenylmethylsulphonyl fluoride - PO peroxidase - rbc red blood cells - RT room temperature - SA Helix pomatia serum agglutinin - TBS Tris buffered saline     

A galactose specific agglutinin, a blood-group H active polysaccharide, and a trypsin inhibitor in albumin glands and eggs of Arianta arbustorum (Helicidae).

Eggs and albumin glands of the land snail Arianta arbustorum contain a powerful agglutinin which reacts specially with rabbit erythrocytes. The agglutination can be inhibited completely by di-, tri-, and oligosaccharides with α-glycosidically (1 → 6) bound galactose residues. β-Linked sugars do not inhibit the agglutinin. The agglutinin activity is not dependent on Ca²⁺ ions. Eggs and albumin glands also contain a blood-group active polysaccharide which, unlike the polysaccharide from the albumin gland of Helix pomatia (Baldo, B. A., and Uhlenbruck, G. 1973. Cross-reactive human blood group H-active polysaccharide from Helix pomatia. I. Detection with catfish anti-H and eel sera. Immunology, 25, 1–13) does not react with anti-H_eel, but does react with the agglutinins of Evonymus europaeus and Laburnum alpinum. The Arianta polysaccharide has been purified and shown to be galactogen. Finally, the occurrrence of a strong trypsin inhibitor has been demonstrated in the extracts of eggs and albumin glands. The inhibitor has been separated by column chromatography. The precipitation lines of both substances have been identified in the immunoelectrophoretogram of the extracts of albumin glands and eggs.     

Binding specificities of the lectins from Helix pomatia, soybean and peanut against different glycosphingolipids in liposome membranes

The binding specificities of the lectins from Helix pomatia, soybean and peanut against glycosphingolipids containing β-linked terminal D-galactose and N-acetyl-D-galactosamine and the role of sialic acid as a modulator of the binding specificity were investigated. The test system used consisted of liposomes containing the glycosphingolipids and lectins coupled to gel columns. Of the investigated glycosphingolipids only ganglioside GM2 bound to soybean agglutinin while Helix pomatia agglutinin was found to bind only GA2. Peanut agglutinin showed good affinity both for ganglioside GM1 and its asialoderivative GA1.     

Molecular properties of the partially SDS-resistant lectin from the albumen gland of Helix pomatia and demonstration of lectin-related molecules on the surface of H. pomatia haemocytes

Lectins (agglutinins) are components of the immunobiologicalrecognition system of vertebrates and invertebrates. The presentstudy focused on the molecular properties of the agglutininfrom the albumen gland of Helix pomatia (HPA) and on the occurrenceof lectin-related molecules on the surface of H. pomatia haemocytes.According to the current model (Hammarström et al., 1972,Scandinavian Journal of Immunology, 1: 259–301), the hexamericHPA of about 79 kDa is composed of three non-covalently associateddimers (26 kDa), each consisting of two disulphide-bridged 13kDa monomers. However, on native-gradient polyacrylamide gelelectrophoresis (PAGE), we obtained high molecular weight bandsrepresenting lectin polymers. The stepwise dissociation of thesewas achieved by incubation with SDS at temperatures from 20to 40°C (1 h) and at 100°C (10 min). The results obtainedon SDS–PAGE included the occurrence of partially SDS-resistanthexamers of about 66 kDa, of two dimer bands of 22 and 19 kDa,and of two minor heteromonomer fractions. Complete dissociationinto heteromonomers of 13 and 11 kDa was achieved by boilingthe lectin (10 min) with SDS under reducing conditions. Fornative lectin molecules, both monomers occurred as disulphide-linkedhomodimers. Monomers or dimers electroeluted from an SDS–gel,reassociated to SDS-resistant oligomers upon re-electrophoresis.Finally, molecules antigenetically related to the lectin wereextracted from the membrane of H. pomatia haemocytes. Anti-HPAantibodies recognized peptides with an apparent molecular weightof about 30 and 56 kDa, which were shown to represent cell-surfacemolecules. (Received 4 March 2008; accepted 9 September 2008)     

Activation of heart contractility of the edible snail <Emphasis Type="Italic">H. pomatia</Emphasis> by thrombin. Study of the role of cAMP

Thrombin acts on mammalian cells through the specific, so-called protease-activated receptors (PARs). The thrombin action is mediated via three out of four known types of these receptors—PAR_1,3,4. Mammalian thrombin receptors, apart from performance of other functions, control cardiac and vascular contractility. It is not known whether receptors of such kind exist in invertebrate animals. In the present work we have showed for the first time that thrombin in the concentration range of 0.01–1 units/ml increases amplitude of contractions of the isolated heart ventricle of the edible snail Helix pomatia. Its effect is reproduced by peptide ligands of receptors PAR₁ and PAR₄ that have sequences Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and Glu-Tyr-Pro-Gly-Lys-Phe (QYPGKF), respectively. A potent activator of cardiac contractility of H. pomatia is serotonin. A comparative study of the mechanisms of action of serotonin and thrombin on the edible snail heart was carried out. cAMP participates in transduction of signal from serotonin receptors. On the membrane preparation from the H. pomatia heart, it was shown that thrombin and peptide ligands PAR₁ and PAR₄, unlike serotonin, did not increase adenylyl cyclase activity. Thus, mechanism of activation of cardiac contractility of H. pomatia by thrombin differs from that of serotonin. It is suggested that molluscs have receptors homologous to protease-activated mammalian receptors.     

Selective in vivo labelling of serotonergic neurones by 5,6-dihydroxytryptamine in the snail Helix pomatia L

1. In the central nervous system of Helix pomatia L. serotonergic neurones were labelled in vivo by injecting 5,6-dihydroxytryptamine into the body cavity of the animals. By the 60th day after the injection, all serotonergie neurones contained a strong, rusty-brown pigmentation located to large lysosomes in the cells.2. The membrane potential, action and synaptic potential generating abilities of the pigmented serotonergic neurones were normal. Their input relations from the cardio-renal system and the lip, respectively, were as intact as in the untreated animals.3. The opportunity to study in vivo labelled and normally functioning neurones may contribute to the better understanding of their function.     

BIOCHEMICAL COMPOSITION OF HELIX SNAILS: INFLUENCE OF GENETIC AND PHYSIOLOGICAL FACTORS

This paper describes the biochemical composition of differentspecies (Helix lucorum, Helix pomatia) and sub-species of snails(Helix aspersa aspersa, Helix aspersa maxima) reared in thesame conditions with a feed (‘Helixal’) speciallydesigned for edible snails. In addition, the composition ofwild H. pomatia and H. lucorum is presented to allow comparisonbetween snails of different origins. Analyses determined thepercentages of proteins, lipids and minerals. They reveal bothsimilarities and differences in composition according to thespecies and the part analysed (whole body, pedal mass, and visceralmass). H. pomatia contains the highest percentage of mineralmatter and the lowest percentage of lipids. Surprisingly, proteincontents are slightly different between artificially rearedH. aspersa maxima of 3 months old and wild H. pomatia. The resultsmake it possible to evaluate nutritional quality of snails withthe composition of the body of four edible snail species. (Received 16 September 1996; accepted 24 May 1997)     

A purification of Helix pomatia alpa amylase involving a novel affinity-binding procedure

Shellfish glycogen was cross-linked by treatment with cyanogen bromide followed by 1,6-diaminohexane. The resulting, insoluble product efficiently adsorbed Helix pomatia alpha amylase [(1 → 4)-α-D-glucan glucanohydrolase] from crude solutions of the enzyme at 0°, but only poorly at higher temperatures. A method was developed for the purification of Helix pomatia alpha amylase involving formation of an enzyme-adsorbent complex in the cold and recovery of the alpha amylase by suspending the washed complex in buffer at 37°. After chromatography of the desorbed alpha amylase on a column of Bio-Gel P-60, the enzyme was homogenous as judged by poly(acrylamide)-gel electrophoresis. An overall purification of 360-fold was achieved with a recovery of 35%.     

Effects of met-enkephalin on electrical coupling between identified neurons in the pulmonate snails,Helix and Lymnaea

1. The effects of met-enkephalin on electrical coupling between molluscan neurons have been investigated using the isolated brains of Helix pomatia and Lymnaea stagnalis.2. In the presence of both serotonin and met-enkephalin, non-rectifying electrical coupling is strongly facilitated between identified respiratory neurons in Helix, whilst coupling between putative, serotonin-containing, ciliomotoneurons in Lymnaea is facilitated by met-enkephalin alone.3. Facilitation of coupling by met-enkephalin is weaker in the strongly coupled neurons, VDl/RPaD2 of Lymnaea.4. These data suggest that met-enkephalin can modulate different groups of electrically coupled cells and may be involved in coordination of motor patterns.     

Studies of lectins XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels

A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (α-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all $\text{N-}\text{acetyl}\text{-α-}\text{d}\text{-galactosaminyl-}$); Ricinus communis, seeds (β-lactosyl-); Onomis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all $\text{α-}\text{d}\text{-galactosyl-}$), Canavalia ensiformis, seeds, (i.e., concanavalin A) ($\text{α-}\text{d}\text{-glucosyl-}$).Physicochemical properties of Glycine soja, Dolichos bifloras, Phaseolus lunatus, Helix pomatia and Ricinus communis lectins correponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physicochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.     

In vitro recalcification of the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The role of the amoebocytes in the calcification process of the shell-repair membrane of the snail, Helix pomatia, was investigated in vitro. The shell-repair membranes were demineralized with 0.5 M EDTA at pH 7.4. For the recalcification of the demineralized membranes two substrates were chosen: (i) Tris-buffered Helix pomatia-saline, pH 7.4, and (ii) Helix pomatia-saline supplemented with 5 mM CaCl₂ and 5 mM NaHCO₃. The membranes were incubated in 2 ml substrate at 37° C and examined after 2 h, 24 h, and 3, 5 and 7 days. Calcium deposition and crystal formation were observed within the membrane incubated in the salt-supplemented substrate. The control membranes were either heat-inactivated or deprived of lipids. No calcium precipitation was observed in control membranes. The experiments show that the recalcification of the shell-repair membrane is under strict cellular control and that the granules released from the amoebocytes serve as sites for calcium deposition. The role of phospholipids in the calcification process is discussed.     

Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils

Summary. The effects of bovine colostrum, absorption of equine colostral immunoglobulins and age on phagocytic and serum opsonizing activity of nine clinically healthy foals were examined. Cells and serum were collected prior to suckling and at 7, 14 and 28 days of age. Seven foals had serum IgG concentrations >600mg/dl whereas two foals had <350mg of IgG/dl. Phagocytic and serum opsonic activity of eight clinically ill foals with <400mg of IgG/dl of serum were also examined before and after plasma transfusion. Phagocytic and serum opsonizing activities were evaluated by an assay for chemiluminescence (CL) after addition of opsonized streptococci. Results showed that bovine colostrum stimulated CL of foal neutrophils. Preliminary characterization of opsonins in bovine colostrum by ammonium sulphate fractionating and heat inactivation indicated that opsonins generating CL were mainly associated with immunoglobulin G. Chemiluminescence generated by foal neutrophils varied with age with foal neutrophils collected at day 14 producing more CL than adult neutrophils ( P <0.05). Foal serum opsonizing activity was similar to adult opsonizing activity if serum IgG concentrations were >600mg/dl but it was less if IgG concentration was <350mg/dl ( P <0.05). Chemiluminescence generated by foal and adult neutrophils was higher when post-transfusion foal serum was used as the source of opsonin than when pre-transfusion foal serum was used ( P <0.05). When adult serum was the opsonin, chemiluminescence of foal neutrophils collected before and after plasma transfusion did not differ. The increase in CL following plasma transfusion was probably due to an increase in serum opsonizing activity.     

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: isolation of a functionally active chemically pure domain and evidence for subunit heterogeneity.

α-Hemocyanin of the vineyard snail, Helix pomatia, is a large oligomer composed of 20 subunits with a molecular weight of 360,000 ± 30,000. Limited proteolysis showed these subunits to be composed of about seven structural domains, each having one oxygen binding site (1). This paper describes the production of these structural domains by tryptic digestion of $\text{1}\text{10}$ hemocyanin molecules. The digestion pattern was followed as a function of time by examining the proteolytically obtained fragments electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels. Based on the molar ratio of each fragment present during digestion the first part of the reaction sequence for trypsinolysis could be deduced. This reaction scheme was simulated by means of an analog computer. Pseudo-first-order reaction rate constants of the various proteolytic cleavages were estimated from the computer generated time course of digestion. On the basis of these results it is postulated that Helix pomatia α-hemocyanin possesses at least two kinds of subunits which differ in their proteolytic susceptibility. These subunits occur in equimolar amounts. A functionally active domain with a molecular weight of about 50,000 has been isolated from a tryptic digest of hemocyanin subunits. This domain seems to be chemically pure, as suggested by the unique sequence of its first six amino acids, viz: Lys-Val-His-Leu-Asn-Lys.     

Isolation of a galactogen utilizing strain of Arthrobacter

The land snail, Helix pomatia, is known to deposit eggs that contain the galactose homopolymer, galactogen. Selective enrichment for galactogen utilizing bacteria in a Helix pomatia habitat resulted in the isolation of a new strain of Arthrobacter. The strain's ability to metabolize galactogen was confirmed by the release of ¹⁴CO₂ from (¹C)-galactogen. The new isolate was able to utilize galactogen, galactose and glucose but not glycogen as sole carbon sources. The type strain A. globiformis ATCC 8010 utilized glucose and galactose, but not galactogen, as carbon sources.     

Opsonic involvement in phagocytosis by mollusk hemocytes.

Macrophages from the gastrophod mollusk Otala lactea are capable of in vitro recognition and phagocytosis of foreign particles such as yeast, mammalian erythrocytes, and bacteria. The degree of intensity of the phagocytic response, in certain instances, is governed by the surface characteristics of the particle in question as well as by the presence of opsonic factors.Hemagglutinins have been implicated as opsonins in certain invertebrates, including mollusks. Otala lacks serum lectins; however, its hemolymph stimulates phagocytosis of formalized yeast but not erythrocytes and bacteria. Hemagglutinin-containing extracts of Otala albumin gland were shown to opsonize formalized red cells. The rate of ingestion of the bacteria used in this study by Otala hemocytes was variable and was not influenced by the presence of hemolymph in the medium.