Isolation and characterization of a lectin from the cephalochordate Branchiostoma lanceolatum (Pallas).

1. A lectin was isolated from an extract of Branchiostoma lanceolatum by affinity chromatography using an asialo-A-peptone-cellulose column. 2 The lectin is a glycoprotein with a carbohydrate content of 2.7%. The mol. wt is 392,000 +/- 28,000. Two subunits of identical size (183,000 +/- 3000) are linked by non-covalent bonds. 3. The lectin agglutinates a variety of erythrocytes including human A, B, O red blood cells as well as human lymphocytes. 4. Hemagglutination activity is inhibited best by N,N',N"-triacetylchitotriose, followed by N,N'-diacetylchitobiose, which is half as inhibitory. 5. Lectin activity is constant between pH 5 and 10. Divalent cations are not required for binding reactions. Activity is totally destroyed by heating to 60 degrees C for 30 min. 6. The lectin is precipitated from the extract by 30-40% ammonium sulfate saturation.     

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.     

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     

Quantification of cytotoxic hemocytes of Mytilus edulis using a cytotoxicity assay in agar

When a thin layer of agar containing a mixture of erythrocytes and Mytilus hemocytes is prepared on slides, the occurrence of plaques of lysed target cells can be observed around a limited number of hemocytes. These hemocytes remain completely intact cells and are viable as evidenced by their ability to phagocytose target cells and/or to form pseudopods. The number of hemocytes releasing cytotoxic molecules has been shown to vary greatly between different animals. The same holds true for the total number of circulating hemocytes, although no correlation exists between the number of hemocytes in the circulation and the percentage of cytotoxic blood cells.     

Origin of a metal-binding protein in serum of Mytilus edulis

The most abundant humoral protein of the haemolymph of Mytilusedulis is known to bind a variety of heavy metals (Renwrantzet al., 1998 Comp. Biochem. Physiol. A, 121: 175–180).This serum protein band 1 (SPB1) was isolated from Mytilus serumby a two-step purification procedure. For the purified proteina molecular weight of 34 kDa was estimated under denaturingconditions and of 37 kDa in its reduced form. After blottingof Mytilus serum onto a nitrocellulose membrane, Cu-bindingby SPB1 was visualized. Furthermore, indications were obtainedof Ca-binding properties of SPB1 and of some bands postulatedto be SPB1-oligomers. Formation of dimers was confirmed by theirreaction with SPB1-specific antibodies. On immunoblots, anti-SPB1detected the metal-binding protein in postnuclear haemocyteextracts. After further fractionation of the cell homogenateby sucrose gradient centrifugation, the indicator antibodiesrecognized SPB1 in the cytosol, as a component of the plasmamembrane and in the fraction of granules which was assumed toinclude secretory vesicles. Investigation of the supernatantfrom a 24 h haemocyte culture indicated release of the metal-bindingprotein by blood-cells. Incubations of different haemocyte monolayerson slides with anti-SPB1 indicated expression of SPB1 in amountsof 55% to almost 100% of the cells, whereby the intensity ofantibody-staining varied between individual haemocytes. Intensitywas independent of cell type as density gradient separationof haemocytes into basophilic and eosinophilic granulocytesresulted in similar staining patterns in both groups. Thus,all granulocytes seemed to be able to produce SPB1 with a varyingdegree of activity indicating a hitherto unknown regulationsystem. (Received 4 June 2007; accepted 6 September 2007)     

Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67–69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Urea-polyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major α-globins and five major β-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical α- and identical β-chains (α₂β₂, symmetric tetramers), or combinations of three (α₂ββ*; αα*β₂) or four (αα*ββ*) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major β-chains could be shown, whereas the α-chains were N-terminally blocked. Accepted: 12 September 1997     

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)     

Heart rate and hemocyte number as stress indicators in disturbed hibernating vineyard snails, Helix pomatia

In humans and other vertebrates, mental or physical stressors may trigger a variety of symptoms generally referred to as the fight/flight response (Cannon 1929). The processes also include variability of the heart frequency as well as leukocytosis. We monitored both body responses in disturbed hibernating vineyard snails, H. pomatia, to obtain information on the stress sensitivity of these “sleeping” invertebrates. The first mild stressor, a 100 meter transport of hibernating snails from the cold room to the laboratory, caused cardiac arrhythmia in the animals. This reaction could have been provoked by mechanical disturbances and/or by raising the body temperature to room temperature. But a change in the ambient temperature did not trigger an abnormal heart rhythm. Different from this observation, we recorded instant heart rate changes in response to knocking on the shell and a very irregular heartbeat occurred when a hole was punched in the shell. With a short time delay upon damaging the shell, a large increase in the number of circulating cells also occurred. This was not observed after knocking on the shell or when snails were adapted to different temperatures for each 48 h. Thus, hibernating snails sense environmental variations which cause an immediate change of the heart frequency and an elevated stimulus level initiates in addition leukocytosis which occurs at a post-stimulus latency. This disparity could indicate the activity of two different stress regulation pathways.Furthermore, the assays demonstrate an increasing linear relation between rising temperature and frequency of heart pulsations (y = 1.01x − 2.1); but the results do not indicate a correlation between heartbeat frequency and the number of cells in circulation. Consequently, neither temperature nor heart frequency seems to influence the number of circulating cells in hibernating H. pomatia.