Immunohistochemical analysis of osteoconductivity of beta-tricalcium phosphate and carbonate apatite applied in femoral and parietal bone defects in rats.

The feature of osteoconductivity, and expression of inductive BMP and transcription factors (Runx2 and Osterix) for osteoblast differentiation, which was related to conductive bone formation, were observed in experimentally created defects in rat femoral and parietal bones filled with beta-tricalcium phosphate (beta-TCP) or carbonate apatite (CAP). Femoral cortical bone defects were repaired by conductive bone formed by osteoblasts differentiated around beta-TCP and CAP, and immunohistochemical observation revealed that the osteoblasts expressed BMPs, Runx2, and Osterix. However, the repair in parietal bone defects was incomplete despite the beta-TCP and CAP filling. Only cells, which differentiated around beta-TCP or CAP, and formed conductive bone expressed BMPs, Runx2, and Osterix. These findings revealed that the osteoconductivity of calcium phosphate materials required the expression of BMPs as the prerequisite for Runx2 and Osterix expression. Therefore, it is suggested that when calcium phosphate ceramics are used as bone substitute materials, BMPs are essential for osteoconductivity.

Phenothiazine and carbazole-related compounds inhibit mitotic kinesin Eg5 and trigger apoptosis in transformed culture cells.

The effects of phenothiazine and carbazole derivatives on the cell-cycle progression of human transformed culture cells were analyzed. After 2 days incubation, 5 microM 1-phenethylamino-3-phenothiazin-10-yl-propan-2-ol (1) induced strong mitotic arrest followed by cell death, and 20 microM 1-(3,6-dichloro-9H-carbazol-9-yl)-3-phenethylamino-2-propanol (5) and 1-(3,6-dibromo-9H-carbazol-9-yl)-3-phenethylamino-2-propanol (6) also induced cell death. The TUNEL-positive nuclei characteristic of apoptotic cell death were detected in cells treated with the compounds. We observed beta- and gamma-tubulins in the arrested cells after the addition of compound 1, and found that more than 90% of the mitotic cells exhibited the monoastral spindle instead of the normal bipolar spindle. The inhibitory effects of compounds 1, 5, and 6 on the microtubule-activated ATPase activity of mitotic kinesin Eg5, which is essential for bipolar spindle formation, were obtained. The most effective inhibitor, compound 1, had an IC(50) of 1.52 microM. We also examined their toxicities on various cell lines. Compound 1 had less toxicity with the non-transformed cell line WI-38, whereas it exhibited strong toxicity with the transformed cell lines WI38VA13, HL-60 and HeLa. On the other hand, a high dose of compound 6 caused cell death in both types of culture cells. These results suggest that compound 1, an Eg5 inhibitor, selectively kills transformed culture cells.

Mutational analysis of growth arrest and cellular localization of human immunodeficiency virus type 1 Vpr in the budding yeast, Saccharomyces cerevisiae.

Viral protein R (Vpr), one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), is responsible for the incorporation of a viral genome into the nucleus upon infection. Vpr also arrests the cell cycle and induces apoptosis in infected cells. Similarly, in yeast, Vpr localizes in the nucleus and shows growth inhibitory activity; however, the molecular mechanism of growth inhibition remains unknown. To elucidate this mechanism, several point mutations of Vpr, which are known to perturb several phenotypes of Vpr in mammalian cells, were introduced in the budding yeast, Saccharomyces cerevisiae. For the first time, we found that growth inhibition by Vpr occurred independently of intracellular localization in yeast, as has previously been reported in mammals. We also identified several amino acid residues, the mutation of which cancels growth inhibitory activity, and/or alters localization, both in yeast and mammalian cells, suggesting the importance of these residues for the phenotypes.

Nordihydroguaiaretic acid, of a new family of microtubule-stabilizing agents, shows effects differentiated from paclitaxel.

Nordihydroguaiaretic acid (NDGA) protected microtubules in NRK cells from depolymerization caused by structurally and functionally diverse drugs such as nocodazole, colchicine, vinblastine, and ilimaquinone. Hitherto reported drugs, although structurally unrelated to paclitaxel, stabilize microtubules in a way similar to that of paclitaxel and compete for paclitaxel binding to tubulin. However, NDGA had activity toward microtubules different from the effects of paclitaxel. In NRK cells, paclitaxel caused microtubule bundle formation in the presence and absence of microtubule-depolymerizing drugs. However, microtubule bundle did not form, and microtubules radiated from the microtubule-organizing center, in cells treated with NDGA. Acceleration of tubulin polymerization in vitro by paclitaxel was strong but that by NDGA was weak. Microtubules polymerized in vitro in the presence of paclitaxel, but not those polymerized in the presence of NDGA, resisted the effects of cold. NDGA seemed to bind to tubulin, but did not compete for [3H]paclitaxel binding to tubulin. These observations indicate that NDGA belongs to a novel family of microtubule-stabilizing drugs.

A novel action of terpendole E on the motor activity of mitotic Kinesin Eg5.

To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.