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Lectin-mediated agglutination of murine lymphoma cells. Cell surface deformability and reversibility of agglutination by saccharides.

Agglutination of S49 mouse lymphoma cells by Ricinus communis I agglutinin can be reversed by the competing haptenic saccharide, lactose, soon after agglutination, but after further incubation in the absence of lectin the agglutination reaction could not be reversed by lactose and the cells remained as multicell aggregates. The irreversibility of S49 cell agglutination was time, temperature and lectin concentration dependent and its onset correlated with ultrastructurally observed deformation of adjacent cell surfaces and an increase in the proportion of adjacent cell surface areas in close apposition within multicell aggregates. Pretreatment of S49 cells with cytochalasin B or cytochalasin B plus vinblastine enhanced R. communis I agglutinin-mediated agglutination, while vinblastine alone and fluoride plus azide had essentially no effect. When drug-treated cells were agglutinated and then incubated in lectin-free drug-containing media for various times prior to lactose addition, the drug effects were more pronounced. Cytochalasin B alone or with vinblastine inhibited lactose reversal of S49 cell agglutination compared to the drug-free controls, while fluoride plus azide enhanced hapten reversibility. Electron microscopic analysis revealed that the onset of agglutination irreversibility correlated with cell surface deformation in the drug-treated cells. Cell aggregates that were more readily reversible by lactose (fluoride plus azide) were unchanged or less deformed, while S49 aggregates treated with cytochalasin B plus vinblastine were more deformed compared to controls without drugs. These experiments suggest a role for cell surface deformability as an important secondary effect during lectin-mediated cell agglutination of S49 lymphoma cells.

Agglutination↗

Terminal saccharides on sperm plasma membranes: identification by specific agglutinins.

Six specific agglutinins were used to identify the terminal sugar residues in the surface oligosaccharides of rabbit and hamster spermatozoa by specific agglutination. Species differences in epididymal sperm were found in the terminal residues, resembling alpha-D-mannose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-galactosamine. Species similarities were found in terminal residues, resembling L-fucose and N-acetylneuraminic acid. When ejaculated rabbit sperm were compared to epididyimal sperm, the latter were more agglutinable with a specific agglutinin recognizing N-acetyl-D-glucosamine.

Acetates↗

Sexual agglutinin of mating-type minus gametes in Chlamydomonas reinhardii. I. Loss and recovery of agglutinability of gametes treated with EDTA.

The effect of EDTA on the mating-type-specific agglutinins located on the flagellar surfaces of Chlamydomonas reinhardii gametes was investigated. The mating-type minus (mt-) gametes lost their agglutinability without apparent loss of motility soon after addition of EDTA at low concentrations (1-2 mM). At the same time, the cells released into the medium agglutinins which can elicit agglutinative responses of mating-type plus (mt+) gametes specifically. When EDTA was neutralized with Mg2+ or removed by centrifugation, the mt- cells quickly replaced agglutinins by protein synthesis: the recovery process was sensitive to cycloheximide, but not to tunicamycin. The EDTA-treated mt+ gametes lost their agglutinins much more slowly than the mt- gametes. The replacement of mt+ agglutinins was inhibited by both cycloheximide and tunicamycin.

Agglutination↗

Mating type-specific cell-cell recognition of Saccharomyces cerevisiae: cell wall attachment and active sites of a- and alpha-agglutinin.

Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2. Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat. The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1. His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed. By site-directed mutagenesis this possibility has now been excluded. With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule. A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations. Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones. Comparisons of the recognition sequences of the S. cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed.

Agglutinins↗

Structure of the Chlamydomonas agglutinin and related flagellar surface proteins in vitro and in situ.

Using the quick-freeze, deep-etch technique, we compare the structure of the cane-shaped plus and minus sexual agglutinin molecules purified from gametes of Chlamydomonas reinhardi. We also describe the structure of three additional gamete-specific fibrillar molecules, called short canes, loops, and crescents, which are structurally related to the agglutinins. Four non-agglutinating mutant strains are found to produce the three latter fibrils but not canes, supporting our identification of the cane-shaped molecule as the agglutinin. The heads of the plus and minus canes are shown to differ in morphology. Moreover, two treatments that inactivate the plus agglutinin in vitro--thermolysin digestion and disulfide reduction/alkylation--bring about detectable structural changes only in the head domain of the cane, suggesting that the head may play an indispensible role in affecting gametic recognition/adhesion. We also present quick-freeze, deep-etch images of the flagellar surfaces of gametic, vegetative, and mutant cells of Chlamydomonas reinhardi. The gametic flagella are shown to carry the canes, short canes, loops, and crescents present in in vitro preparations. The cane and crescent proteins self-associate on the flagellar surface into stout fibers of uniform caliber, and they align along the longitudinal axis of the flagellum. The short canes and loops co-purify with flagella but, in the presence of mica, dissociate so that they lie to the sides of the flagella. The agglutinin canes of both mating types are oriented with their hooks at the membrane surface and their heads directed outward, where they are positioned to participate in the initial events of sexual agglutination.

Agglutinins↗

Isolation of cold agglutinins in Eperythrozoon suis-infected pigs.

A simple and reliable procedure is described for isolating the cold agglutinins associated with Eperythrozoon suis-infection in swine. After initiating microagglutination by cooling the blood samples, the erythrocytes were separated by density-gradient centrifugation using Ficoll-Paque, resuspended in RPMI-medium and warmed to 40 degrees C. The cold agglutinins could be removed from the supernatant by a subsequent centrifugation. Double immunodiffusion, polyacrylamide gel electrophoresis and immunoblotting showed that the cold agglutinins isolated consisted of IgM antibodies exclusively. Their ability to induce agglutination in cooled erythrocytes from healthy pigs confirmed that they were genuine cold agglutinins. The method paves the way for more detailed investigation into the mechanisms of this agglutinating disease.

Agglutination↗

Red cell destruction in cold agglutinin disease.

OBJECTIVE: The severity of autoimmune hemolytic anemia (AIHA) caused by cold agglutinins (CAs) is known to differ markedly in chronic CA disease as well as in postinfection cold agglutination. The various cold agglutinin specificities and mechanisms of red cell destruction are described. DATA SOURCES: Original papers and reviews of the German and English literature (Medline research); own results. SELECTION CRITERIA: Original papers and reviews of the recent years on studies of the biochemistry and specificity of CAs and the pathophysiology of AIHA. RESULTS: A crucial point for the severity of AIHA caused by CAs is the CA-binding capacity to red cells in vivo. This is reflected by the serologic behavior of the actual CA in vitro, represented by its thermal amplitude. Because most CAs are IgM molecules, red cell destruction by CAs is limited to mechanisms initiated by complement (C) activation. In recent years, several CA specificities, in addition to anti-I/i, have been identified on a serological and biochemical basis. CAs of the IgG and rarely of the IgA isotypes have been found. CONCLUSION: The CA-induced red cell destruction does not only depend on CA titer or its thermal amplitude. The severity of CA-induced AIHA may also depend on CA isotype and/or specificity. Therefore, the complement activation capacity of CAs with a given isotype but different specificities has to be elucidated in further studies.

Agglutinins↗

Shedding of Treponema hyodysenteriae, transmission of disease, and agglutinin response to pigs convalescent from swine dysentery.

Fecal shedding of Treponema hyodysenteriae, transmission of disease, and humoral antibody production against the large spirochete were evaluated in pigs convalescent from experimentally induced swine dysentery. Untreated pigs (n = 21) and 5 pigs that had been treated with virginiamycin were included in the study. Treponema organisms were culturally detected in the feces of 2 untreated pigs as long as 70 and 71 days, and in the feces of 1 treated pig as long as 83 days after the last clinical evidence of disease. Of 8 convalescent pigs that intermittently discharged T hyodysenteriae in their feces, 4 transmitted clinical disease to exposed susceptible pigs. One of the convalescent animals has been free of clinical signs of disease for 57 days before introduction of the susceptible pigs. Treated and untreated convalescent pigs developed similarly elevated agglutinin titers that were maintained as long as 150 days after infection. There was no apparent correlation between the frequency or duration of fecal shedding of the spirochetes and the magnitude of the agglutinin titers of the convalescent pigs.

Agglutination Tests↗