Agglutinin Activity Research Articles

Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization.

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.

An esterase isozyme in non-hemoglobin proteins of cell lysate reacting with mushroom lectin.

Of 20 kinds of plant lectins examined, agglutinin activity of only mushroom lectin was inhibited with non-hemoglobin proteins of the red cell lysate. Chromatography of non-hemoglobin proteins on DEAE-cellulose and on hydroxylapatite resulted in isolating a protein which gave a single precipitation line with esterase activity in immunoelectrophoresis. SDS-polyacrylamide gel disc electrophoresis demonstrated that the protein consisted of two subunits molecular weight of which was approximately 100,000 each.

Respiratory tularemia: comparison of selected routes of vaccination in Fischer 344 rats.

Fischer 344 rats were given the attenuated live vaccine strain of Francisella tularensis by small-particle aerosol, intranasal instillation, or intraperitoneal, intramuscular, or subcutaneous injection. All of the vaccinated rats developed subclinical infection by 3 days after exposure, which cleared by day 28. Temporal patterns and concentrations of the live vaccine strain organism within the hosts were dependent on the route of vaccination. Pathological alterations were limited to minimal lung lesions in aerosol-vaccinated rats and mild splenitis in intraperitoneally vaccinated rats. Agglutinins to live vaccine strain were detected in the serum of each vaccinated animal and in the bronchoalveolar wash fluids of 66% of the aerosol-vaccinated rats. Agglutinin activity of the vaccinated rats was associated predominantly with the immunoglobulin M class. Regardless of the route of vaccine administration, all vaccinated rats survived an aerosol challenge of 5.3 log10 cells of virulent F. tularensis, whereas all nonvaccinated rats died. Systemic infection did not occur in the vaccinated rats. Pulmonary infection was not prevented in the vaccinated rats after aerosol challenge, but proliferation of the virulent F. tularensis organisms in the lungs was significantly lower (analysis of variance, P less or equal to 0.01) than that which occurred in the control animals. These studies demonstrate the utility of the inbred Fischer 344 rat as a model host for further investigations of F. tularensis infection and its associated immune response.

Composition and immunochemical properties of glycoproteins with anti-B agglutinin activity isolated from Euonymus sieboldiana seeds.

Five major glycoproteins with anti-B agglutinin activity were isolated from seeds of Euonymus Sieboldiana by a procedure based on precipitation with ammonium sulphate, Sepharose 4B gel filtration, CM- and DEAE-Sepharose chromatography and Sephacryl S-200 gel filtration. The purified glycoproteins each gave a single symmetrical peak on Sephacryl S-200 gel filtration with elution volumes corresponding to molecular weights of approximately 15,000 to 130,000, each forming a single precipitin line on gel diffusion plates with anti-E. Sieboldiana antibody. These anti-B glycoproteins were rich in acidic amino acids without cysteine and methionine and contained about 8--36% carbohydrate, of which galactose, arabinose and glucose were the predominant sugars, with small amounts of glucosamine, rhamnose, fucose, xylose, mannose and ribose. The most anti-B active lectin agglutinated human B red blood cells at a concentration of 2 micrograms/ml and was strongly inhibited by melibiose. The three other lectins with anti-B agglutinin activity, however, were not inhibited by any galactose-containing glycosides.

An evaluation of the specificity of salivary agglutinins.

Four bacterial strains collected from tooth surface, oral buccal mucosa, skin of the upper lip, and rectum were used to evaluate the specificity of salivary agglutinins. A saliva sample was absorbed with each of the strains, and the remaining supernatants were tested for agglutinin activity against all strains. The effect of concanavalin A and pH on the S. mutans and S. mitior agglutinins was studied. The conclusion was drawn that the agglutinins for the S. mutans and S. mitior strains differed but that the S. mitior strain rested carried a receptor site for the S. mutans agglutinin without becoming agglutinated. The S. mutans agglutinin seems to have either unsubstituted mannopyranosyl or N-acetylated glucopyranosyl residues as structural components.

Agglutinating Antibody to Aeromonas hydrophila in Wild Largemouth Bass

Abstract Among largemouth bass Micropterus salmoides in Par Pond, South Carolina, a significantly larger percentage of those with red-sore disease were positive for anti-Aeromonas hydrophila agglutinin than of uninfected fish. Highest titers occurred during summer and fall, when the prevalence of the disease was declining. Most agglutinin activity was associated with a single serum fraction; the agglutinin has an apparent molecular weight of >340,000 daltons, suggesting it may be a macroglobulin-like antibody. Homologous agglutinin reacted better with A. hydrophila than heterologous agglutinin. Differences in severity and duration of red-sore epizootics in the southeastern United States may be due to differing virulence among strains of A. hydrophila.

The molecular mechanism of cryoprecipitation and cold agglutination of an IgM lambda Waldenström macroglobulin with anti-Gd specificity: sedimentation analysis and localization of interacting sites.

The physicochemical properties and immunologic specificity of an IgM lambda cryoglobulin with cold agglutinin activity (designated MAT) were investigated. Discrete binding sites involved in cryoprecipitation were shown to exist on the reductive 7S subunits and tryptic Fab mu and Fc mu 5 fragments derived from MAT. Analytical ultracentrifugation indicated the nonprecipitating MAT subunits self-associated into rapidly sedimenting paucidisperse boundaries of 32S, 25S, and 8.7S at 13 degrees C, which were converted to a monodisperse 5.7S boundary at 35 degrees C. Furthermore, MAT Fab mu and Fc mu 5 fragments exhibited association with each other at 13 degrees C but not at 35 degrees C when studied by analytical ultracentrifugation. The cold agglutinin activity of MAT can be inhibited by sialyllactose or treatment of erythrocytes with neuraminidase but not papain. Therefore, the membrane determinants of human erythrocytes are Gd (glycolipid-dependent or protease-resistant glycoproteins) with terminal sialic acid. Treatment of MAT with neuraminidase abolished its precipitating properties, but had no effect on its ability to agglutinate untreated erythrocytes. The cryoprecipitation and cold agglutination phenomena exhibited by MAT therefore seem to involve identical or cross-reacting carbohydrate antigens with terminal sialic acid residues.

Fibrinogen is the receptor for the endogenous lectin of human platelets.

Washed platelets activated by alpha-thrombin, gamma-thrombin, thrombocytin or the ionophore A23187 (ref. 3) lose their disk shape, produce pseudopodia and become cohesive. This cohesiveness is accompanied by the expression of an endogeneous haemagglutinin which, although apparently bound to the platelet membrane, is dependent on cell secretion. The interaction of this agglutinin with appropriate receptors on other platelets is believed to be responsible for aggregation. We report here that platelets can be prepared which lack agglutinin activity but have receptor function, that afibrinogenaemic platelets lack receptor activity, and that fibrinogen is the receptor for the agglutinin secreted by activated platelets.

Spurious macrocytosis, a common clue to erythrocyte cold agglutinins.

In four patients with cold agglutinins, erythrocyte mean corpuscular volume was increased. Erythrocyte volume histograms showed that in each case macrocytosis was due entirely to doublet erythrocytes counted as single cells. For two of the four patients, macrocytosis was reported intermittently, when the blood had been allowed to cool to room temperature during laboratory processing. For these two patients, macrocytosis due to doublet erythrocytes could be elicited in vitro proportionate to the degree of cooling of the blood and could be abolished to rewarming to 37 C. Two other patients had macrocytosis constantly during the acute illness. Otherwise unexplained macrocytosis may reflect cold agglutinin activity without overt hemolysis, particularly if the macrocytosis is intermittent.

Mice refractory to lipopolysaccharide manifest high immunoglobulin A responses to orally administered antigen.

Lipid A-nonresponding C3H/HeJ mice manifested high immune responses to orally administered (either by feeding or by intragastric immunization) heterologous erythrocytes when compared with syngeneic lipid A-responding C3H/HeN mice. Prolonged consumption of horse erythrocytes resulted in a significant secretory immune response in both C3H mouse strains as evidenced by high salivary agglutinin titers. Although salivary agglutinin titers were only slightly greater in C3H/HeJ mice than those of C3H/HeN mice, serum agglutinin titers and immunoglobulin A (IgA) levels were consistently higher (two- to fourfold) in C3H/HeJ mice. The appearance of anti-horse erythrocyte plaque-forming cell responses in spleens of immunized animals was followed by an increase in salivary anti-horse erythrocyte agglutinin activity. Peak levels of both responses were attained after approximately 3 weeks of immunization. Differences in immune responsiveness between C3H mouse strains were also evident at the cellular level since splenic IgA anti-horse erythrocyte plaque-forming cell responses in fed C3H/HeJ mice were twofold higher than those in similarly treated C3H/HeN mice. This higher response pattern was also observed when C3H/HeJ mice manifested threefold higher splenic IgM and IgA plaque-forming cell responses to intragastrically administered sheep erythrocytes. Thus, higher responsiveness was observed in the C3H/HeJ mice given heterologous erythrocytes by the oral route. Furthermore, levels of serum IgA in 10- to 12-month-old nonimmunized C3H/HeJ mice were higher than those of C3H/HeN mice. These findings suggest that lack of host responsiveness to lipopolysaccharide affects the manifestation of subsequent immune responses to orally administered antigens. The possible mechanisms and implications of this high responsiveness are discussed.

マガキ組織における凝集素と殺菌素の活性

High natural agglutinin activities against sheep erythrocytes were observed in the extracts of the gill (mean titer: 141) and digestive diverticula (DD, mean titer: 128) from adult oyster, Crassostrea gigas, under hanging culture. Lower but considerable activities were found in the extracts of the mantle and adductor muscle. The natural bactericidal activity against HS 29 strain (Arthrobacter sp.) was conspicuously high in the DD extracts (mean titer: 231) and detectable also in the mantle and gill. The natural agglutinin activites of DD and gill declined sharply with progressive develop ment of the gonad and was minima just before and during the spawning, However, these activities showed a rapid rising trend after the spawning season. The agglutinin activities of DD and gill were enhanced by the injection of 0.1ml/oyster of 10% sheep erythrocyte suspension into the connective tissue surrounding DD. The bactericidal activity of DD was observed to increase after the injection of formalin-killed HS 29 strain vaccine which was given into the connective tissue surrounding DD (dose: 1.8×108 cells/g fresh soft-body wt.) or the adductor muscle (8.3×105 cells/g fresh soft-body wt.).

軟体動物の生体防御機構 Ｉ ホタテガイ組織における殺菌素と凝集素の活性

The extracts of the digestive diverticula (DD) from the normal two-year-old scallop, Patinopecten yessoensis, in hanging cultures were found to possess a low level of natural bactericidal activeity (bactericidin titer: 5 to 10) against HS 29 strain, Arthrobacter sp. No activity, however, was observed in the extracts of the kidney, mantle and gill. The natural agglutinin activity against sheep erythrocytes was extremely high (agglutinin titer: 128) in the DD extracts, as compared with that in the extracts of the gonad, giil and adductor muscle. The natural bactericidal activity of DD which was maximal from March to May declined with the rise of water temperature in the vicinity where scallops were cultivated, and was minimal in hot summer. It exhibited a recovery trend after summer and attained its maximum level in December. The seasonal changes paralleled those in the natural agglutinin activity of DD. The baseline killing power of DD for HS 29 strain was enhanced conspicuously by admi-nistration of HS 29 strain vaccine (formalin-killed, ca. 1×107cells/ml). The duration of the enhancement was approximately 5 days at 10°C.

A lectin which binds specifically to beta-D-galactoside groups is present at the earliest stages of chick embryo development.

Extracts obtained from chick embryos at the pre-gastrula and gastrula stages are able to agglutinate trypsinized rabbit erythrocytes fixed with glutaraldehyde. Agglutination is inhibited by saccharides sharing a beta-D-galactopyranoside configuration. Agglutinin activity is also inhibited by desialysed fetuin which bears terminal galactose residues but not by native fetuin, desialysed agalacto-fetuin, bovine submaxillary mucin and desialysed bovine submaxillary mucin. Lectin activity is present in extracts obtained from the embryonic area of the blastoderms as well as in extracts from extra-embryonic endoderm and ectoderm. In extracts subjected to gel filtration on ECD Sepharose, lectin activity eluted between ovalbumim (mol. wt 45 000) and chymotrypsinogen (mol. wt 25 000). Under some experimental conditions, thiodigalactoside, the most potent inhibitor of lectin activity, inhibited the aggregation of cells of the extra-embryonic endoderm of the primitive chick embryo.

Partially purified antibodies used in a solid-phase radioimmunoassay for detecting candidal antigenemia

The development of a solid-phase radioimmunoassay procedure for the detection of Candida albicans antigens in serum of mice is described. Antibodies against C. albicans that were used in the radioimmunoassay procedure were partially purified from immune serum by a C. albicans antigen-coupled affinity column. Elution of anti-C. albicans antibodies from the column was by glucose and mannose; 4 mg of protein was recovered per ml, which contained 50% of the candidal agglutinin activity of immune serum. Also, 81% of the protein (partially purified antibody) recovered was adsorbed by whole C. albicans cells. Anti-C. albicans antibodies were either coupled to Sepharose 4B for use as the solid phase to bind candidal antigen in serum of infected animals, or radioiodinated (125I) for use as a tracer molecule to bind to the candidal antigen solid-phase complex. Although control experiments indicated that at least 100 ng of candidal antigen should be present in a serum specimen for a positive radioimmunoassay test, candidal antigenemia was detected in 70.4% of infected mice even in cases where blood cultures for C. albicans were negative. With further refinement and adaptability to human serum, the radioimmunoassay test may become a helpful tool for use in the diagnosis of systemic candidiasis.

On the receptors of human red cells reacting with Phaseolus coccineus L. lectin.

In order to elucidate receptors of proteolytic enzyme-treated red cells which react with Phaseolus coccineus L. lectin, the receptors prepared by affinity chromatography were serologically investigated. P. coccineus lectin had high agglutinin activity for bromelin-, papain- and pronase-treated red cells but that for the cells treated with ficin and trypsin was relatively low. Analyses of chemical composition revealed that sialic acid of the receptors from normal red cells was considerably much as compared with that from the treated cells. On the contrary, the enzyme treatment did not affect particularly carbohydrate composition of the receptors. Disc electrophoresis showed that the patterns of receptors from red cells treated with bromelin or papain were different from those from the other cells. On two-dimensional immunoelectrophoresis, the receptor of trypsin-treated cells gave five precipitation lines against anti-stroma and that of papain-treated cells three lines, but any other receptors showed no line. These findings indicate that there are plural receptors for P. coccineus lectin in red cells treated with each of proteolytic enzymes and that the receptors from respective red cells have electrophoretically and serologically different property.

Studies on secretor specific H antigen of water-soluble blood group substances. I. Findings of anti-H(Se) precipitin in eel sera.

The presence of two kind of precipitins against blood group H substances from the human stomach and ovarian cyst was demonstrated in eel (Anguilla japonica) sera by immunodiffusion techniques in agar gel. One of them which forms the precipitation line to antigen side is the known anti-H precipitin containing anti-H agglutinin activity. The precipitating activity of the eel anti-H serum with H substance was inhibited by L-fucose and 2'fucosyllactose. The anti-H precipitin also reacts well with H substance from the hog stomach, but does not show clear precipitation lines with human secretor salivas and 1% solution of preparations of H substances from the small intestine, meconium and semen. Another precipitin which forms a precipitation line to the antiserum side in agar gel reacts well with H substances from human secretions and tissues. Because this precipitin reacted with water-soluble substances from human secretor, the antigen was named H(Se) and antibody anti-H(Se). The anti-H(Se) precipitin is not absorbed with group O red cells and the H and H(Se) activities of H substance against eel precipitins are decomposed by α-(1→2)-fucosidase.

Lactose sensitive lectin of chick retina and spinal cord

A factor extracted from chick embryo retina and spinal cord was found to agglutinate rabbit erythrocytes in vitro . The amount of agglutinin activity varies markedly during embryonic development in the spinal cord, rising to a peak of 7692 units/mg protein at 10 days and then falling to 1120 units/mg protein in the spinal cords of newly hatched chicks. Agglutination activity of the retina developed later (zero at 8 days) and after day 16 plateaus at values 10% that of the peak spinal cord activity. Despite such marked differences in hemagglutination activity and the pattern of development, both the retina and spinal cord lectins were blocked by the same saccharides with essentially identical potency ratios. Lactose (4-o-β-D-Galactopyranosyl-D-glucose) was the most potent inhibitor and was 1,000 times more potent than melibiose (6-o-α-D-Galactopyranosyl-D-glucose).

A glactose 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 Ca 2+ 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.

Lobster ( Homarus americanus) hemocytes: Classification, differential counts, and associated agglutinin activity

Four hemocyte types were recognized in the lobster based on size and refractile nature of the granules, the ratios of cytoplasm to nucleus, and Giemsa stain characteristics. Two hyaline types were designated as prohyalocytes (1.8%) and hyalocytes (64.2%), and two granular types were termed eosinophilic granulocytes (12.2%) and chromophobic granulocytes (21.9%). There was no significant difference in the percentages of the different hemocyte types (differential hemocyte counts) between sexes, but hyalocyte and eosinophilic granulocyte percentages varied significantly between populations of lobsters. The data suggested that the difference in agglutinin activity (HA) between lobsters with the same total hemocyte numbers was due to activity associated with fixed hemocytes or quantitative differences in HA activity associated with one or more hemocyte types, rather than an increase in the percentage of any one particular type in circulation.

Discovery of a Lectin with Affinity for the HBs Antigen

An extract of marguerite leaves was found to contain an agglutinin which has affinity for HBs (adr type) antigen-sensitized sheep erythrocytes and for pure HBs antigen. This agglutinin activity was inhibited by the plasma of hepatitis patients who were known to have circulating HBs antigen. The agglutinin activity was also inhibited by 0.03M N-acetylneuraminic acid.