Bicyclic and tricyclic C–C mismatch-binding ligands bind to CCG trinucleotide repeat DNAs

A structural change-inducible ligand that binds to CCG trinucleotide repeats was developed via bivalent interaction and enlarging the aromatic ring system.

ANALYSIS OF EFFECTIVENESS OF USING IgY FROM CHICKEN IN SANDWICH METHOD OF HBsAg TESTING

Aim. Study antigen-binding ability of polyclonal antibodies (PCA) of chicken compared with monoclonal antibodies (MCA) of mice in the model of interaction with HBsAg. Materials and methods. Mice MCA 18C8 and MKA F3/F4 (IgG) were used, effective in enzyme immunoassay sandwich method of HBsAg determination (with a minimal detection dose of 0.017 ng/ml), and affinity purified anti-HBsAg PCA of chicken (IgY), obtained from 2 immunized birds (PCA No. 1 and PCA No. 2). The ability of antibodies to bind HBsAg was evaluated by analytical sensitivity (slope of binding curve) of solid-phase enzyme immunoassay system using mice MCA and chicken PCA. Results. PCA No. 2 has provided a statistically significant 40% increase of analytical sensitivity, compared with «standard» immobilized MCA 18C8, in model experiments of binding of peroxidase-labeled HBsAg. However, transition from model experiments to use of PKA No. 1 and PICA No. 2 in sandwich method of determination of HBsAg instead of immobilized MCA 18C8 or detecting MCA F3/F4 in all the cases, on the contrary, resulted in a decrease of analytical sensitivity. Conclusion. A lower flexibility of chicken PCA was assumed to be able to impede bivalent interaction in sandwich method, resulting in formation of less stable immune complexes. Without challenging value of IgY for the creation of immunochemical diagnostic methods, these facts and assumptions indicate a necessity of a deeper elucidation of the best areas of their application.

Plasminogen binds to plasmin-modulated factor Xa by Ca2+- and C-terminal lysine-dependent and -independent interactions

SummaryPlasminogen binding to receptors involves both C-terminal lysine-dependent and -independent interactions. The latter are poorly understood. Our earlier work demonstrated a novel Ca2+-enhanced bivalent interaction between plasmin-cleaved FXa (FXa33/13) and plasminogen truncated at Lys78 (Lys-Pg). Here we hypothesized that the effects of Ca2+may enable dissection of the C-terminal lysine-dependent and -independent interactions. To evaluate the role of the Glu-plasminogen (Glu-Pg) amino acids 1–77, binding of FXa33/13 to immobilized Glu-Pg was compared to Lys-Pg by surface plasmon resonance. Under identical conditions, approximately half the amount of FXa33/13 bound to Glu-Pg. The simplest fit of data suggested a 2:1 plasminogen:FXa33/13 stoichiometry for both, which were proportionately enhanced by Ca2+. Only Lys-Pg demonstrated significant Ca2+-independent binding to FXa33/13. In the presence of Ca2+, weak C-terminal lysine-independent binding could be detected, but only for Glu-Pg. The elastase-generated plasminogen fragment encompassing the angiostatin-like kringle domains 1 to 3 (K1–3) inhibited binding of FXa33/13 to Lys-Pg, whereas fragments corresponding to kringle 4- and kringle 5-protease domain had no effect. Immobilized K1–3 binding to FXa33/13 had both Ca2+-dependent and -independent components. The principal KMd for the interaction was 10-fold higher than Lys-Pg. In the presence of Ca2+, eACA inhibited FXa33/13 binding to K1–3 by 30%, but eliminated binding in the absence of Ca2+. These studies suggest that Ca2+-dependent and -independent binding of Lys-Pg to FXa33/13 are C-terminal lysine-dependent. The N-terminal 1–77 amino acids of Glu-Pg confer significant C-terminal lysine-independent binding, which may play a role during the initiating stages of plasminogen activation.

Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells.

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.