Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

A killer factor produced by the cellular slime mold Polysphondylium pallidum

A killer strain was discovered in cellular slime molds. The wild isolate CK-8 of Polysphondylium pallidum kills all other strains in Polysphondylium and Dictyostelium, as far as could be determined, except strain CK-8 itself and its complementary mating type strain CK-9. Growth-phase cells of CK-8 excrete a killer factor which is sensitive to heat, above 60°C for 5 min, and trypsin. The apparent molecular mass of the factor was determined at 10 000–12 000.Abbreviations BSS Bonner's salt solution - CM conditioned medium     

Calcium-dependent regulation of actin filament bundling by lipocortin-85

Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels.     

Bsr-REMI: An improved method for gene tagging using a new vector inDictyostelium

Using a plasmid pBsr2 which carries a blasticidin S-resistant gene, we have improved the method of REMI (restriction enzyme-mediated integration) provided for insertional mutagenesis inDictyostelium discoideum (bsr-REMI). To confirm usefulness of thebsr-REMI, transformation efficiency, copy number of integrated DNA, and randomness of integration into genome were examined.     

Involvement of Cell Surface Carbohydrates in the Sexual Cell Fusion of Dictyostelium discoideum

In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum ), monoclonal antibodies were raised against partially-purified gp 70, a fusion-related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion-blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N-linked glycosylation of proteins in cell fusion. All above results suggest that N-linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum .     

Cell-division inhibitor produced by a killer strain of cellular slime mold Polysphondylium pallidum

The killer strain CK-8 of cellular slime mold Polysphondylium pallidum produces a cell-division inhibitor, in addition to a killer factor. This inhibitor represses cell division of many strains and species of cellular slime molds, except CK-8 itself and its complementary mating-type strain. It is sensitive to both heat and trypsin, and capable of binding to Con A. Its apparent molecular mass is more than 100 kDa. Repression of cell division by this inhibitor is reversed by trypsin treatment of the cells.     

Isolation of a Spore Germination Inhibitor from a Cellular Slime Mold Dictyostelium discoideum

The functional properties of gluten obtained by treating with chymotrypsin at alkali pH were investigated. The gluten was treated by chymotrypsin at pH 10.0 and 20°C, and was found to be deamidated to a state that was scarcely subject to proteolysis by chymotrypsin. The degree of deamidation of the gluten reached about 25% by this treatment for 2 hr. The functional properties of the gluten thus obtained were investigated in regard to deamidation. The enzymatically deamidated gluten greatly improved such functional properties as solubility and emulsifying ability. In particular, the solubility of the treated gluten was remarkably high in the pH range of 5 to 8, in which native gluten is insoluble. It was apparent that the improvement in functional properties of gluten was mainly due to the deamidation induced by treating with chymotrypsin at pH 10.0 and 20°C.     

Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity

Photosynthesis Research - X-ray crystallographic analysis (1.9-Å resolution) of the cyanobacterial photosystem II (PSII) dimer showed the presence of five phosphatidylglycerol (PG) molecules...     

Liposome-encapsulated superoxide dismutase suppresses liposome-mediated augmentation of TNF-alpha production from peripheral blood leucocytes

Liposome-encapsulated hemoglobin (LEH), a candidate for a red cell substitute, has been reported to be cleared from circulation primarily by the phagocytic system and modulate the production of inflammatory cytokines, such as TNF-alpha and IL-6, both in vivo and in vitro. In this study, we investigated the effects of liposome vesicles on the LPS-induced TNF-alpha production using a whole blood culture system. We also studied the effects of superoxide dismutase (SOD) encapsulated in liposome on the cytokine production. The pre-treatment of whole blood with liposome vesicles potentiated the LPS-induced TNF-alpha production. The encapsulation of SOD in the liposome vesicles suppressed the liposome-mediated augmentation of TNF-alpha production in a dose-dependent manner. These results suggest that encapsulation of SOD in LEH decreases the production of inflammatory cytokines from the phagocytic system which may be caused or augmented by LEH infusion in vivo.