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The effect of magnesium and zinc on the growth kinetics of a hydroxyapatite (0001) face in pseudophysiological solutions was investigated. The growth rates of the (0001) face were measured under various concentrations of magnesium or zinc using Moire phase shift interferometry coupled with surface observation by atomic force microscopy. The (0001) face grew not in a spiral growth mode but in a multiple two-dimensional nucleation growth mode. It was shown that the lateral growth of two-dimensional islands on the (0001) face was inhibited by the addition of magnesium or zinc, following an inhibition of the normal growth rate of the face. Although both cations inhibited growth, zinc was found to reduce the growth rate about 1000 times more effectively than magnesium.

We have developed a novel system for expressing recombinant actin in Dictyostelium. In this system, the C terminus of actin is fused to thymosin ␤ via a glycine-based linker. The fusion protein is purified using a His tag attached to the thymosin ␤ moiety and then cleaved by chymotrypsin immediately after the native final residue of actin to yield intact actin. Wildtype actin prepared in this way was functionally normal in terms of its polymerization kinetics and muscle myosin-mediated motility. We expected that this system would be particularly useful for expressing toxic actin mutants, because the actin moiety of the fusion protein is unlikely to interact with the actin cytoskeleton of the host cells. We therefore chose to express the E206A/R207A/E208A mutant, which appears to be dominant lethal in yeast, as a model case of a toxic actin mutant that is difficult to express. We found that the E206A/R207A/E208A mutant could be expressed and purified with a yield comparable to the wild-type molecule (3-4 mg/20 g cells), even though green fluorescent protein-fused actin carrying the E206A/ R207A/E208A mutation was expressed at a much lower level than wild-type actin. Purified E206A/R207A/E208A actin did not polymerize, even in the presence of muscle actin; however, it accelerated polymerization of muscle actin and inhibited the nucleating and severing activities of gelsolin. Given that the location of the substituted residues is near the pointed end face of the mutant, we suggest that E206A/R207A/E208A actin behaves like a weak pointed end-capping protein that perturbs the actin cytoskeleton of the host cells.Actin filaments in live cells undergo continuous, dynamic turnover and remodeling. These processes involve polymerization, depolymerization, severing, capping, and branching of actin filaments through interaction with a vast array of actinbinding proteins. At present, understanding how the activities of these proteins are regulated is a major issue in cell biology. In addition, it is known that subunits within actin filaments are able to take different conformations and that, in certain cases, large blocks of subunits spanning long distances within individual filaments undergo cooperative conformational changes (1-6). For instance, in the case of thin filaments in striated muscle, Ca 2ϩ -triggered conformational changes in the filaments are enhanced by cooperative conformational changes induced by myosin (reviewed in Ref. 7). In other cases, however, the physiological function of cooperative conformational changes in actin subunits remains unclear. Thus, a number of interesting questions as to the structure and function of actin filaments remain unanswered. Dominant negative actin mutations have been identified from genetic screens, and these mutants may be useful for the elucidation of actin functions, because the use of dominant negative mutant proteins often provides a unique means of dissecting the molecular mechanisms underlying complex phenomena, particularly those that involve direct interaction among ...

Using ab initio methods, a single isomer of D 3h symmetry was found to be preeminent on the Ca 3 (PO 4 ) 2 cluster potential energy surface. This isomer has the distinct feature that each calcium atom is surrounded by four oxygen atoms. We studied the aggregation of this isomer in the [Ca 3 (PO 4 ) 2 ] n system with n being 2 or 3. This gives us an energy reference for analyzing the stability of the so-called Posner's cluster Ca 9 (PO 4 ) 6 which is the core of the actual structural model of amorphous calcium phosphate. Posner's cluster is shown to be significantly stabilized in comparison to the energy reference.