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In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Rat peritoneal macrophages were incubated in the presence of cycloheximide or dexamethasone to inhibit the induction of cyclooxygenase (COX)-2 protein synthesis. Thereafter, when the macrophages were incubated in the presence of arachidonic acid, PGE2 production was increased. Western blot analysis demonstrated that COX-2 protein levels were low and were not affected by arachidonic acid treatment. COX-1 protein levels were not affected by arachidonic acid treatment either. The COX-2 inhibitors NS-398 and nimesulide only slightly inhibited PGE2 production, whereas the COX-1/COX-2 inhibitors indomethacin, piroxicam and tenoxicam strongly inhibited PGE2 production. This suggests that under these conditions, PGE2 production is dependent on COX-1. After the macrophages were treated with aspirin to inactivate existing COX-1 and COX-2, however, treatment with 12-0-tetradecanoylphorbol 13-acetate increased PGE2 production. Furthermore, COX-2 protein levels were markedly increased by 12-0-tetradecanoylphorbol 13-acetate treatment, whereas COX-1 protein levels did not change. In this case, both the COX-2 and the COX-1/ COX-2 inhibitors inhibited PGE2 production. This suggest that under these conditions, PGE2 production is dependent on COX-2. Effects of auranofin on COX-1-dependent and COX-2-dependent PGE2 production were examined. We found that auranofin stimulated COX-1-dependent PGE2 production but inhibited COX-2-dependent PGE2 production in a concentration-dependent manner. The latter effect was found to be due to the inhibition of COX-2 protein induction. These findings might explain the mechanism of the antirheumatic and anti-inflammatory activities of auranofin.