The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH2-terminal EC1 domain in the homophilic ...binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of ∼64 μM. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH2-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions. We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.
In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular ...calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.
A three-dimensional (3D) finite-element formulation for calculating Meissner currents in multiply connected superconductors is presented. The fluxoid quantization condition is ensured as simply as ...possible. The problem is formulated so that we have to solve two systems of equations by the use of a conjugate gradient algorithm without preconditioning. Meissner currents and magnetic-flux density are numerically evaluated in a superconducting tube and around a vortex. These results are compared with analytical solutions.
A three-dimensional (3-D) finite element formulation for calculating Meissner currents in superconductors is presented. The authors have chosen a magnetic vector potential formulation, which also ...enables them to simulate ferromagnetic shielding. The equations are written so that the problem can be solved by the use of a conjugate gradient algorithm without preconditioning. Numerical results on normal-superconductor junctions and on superconducting lines are compared with analytical solutions.
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