Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.

Excess cyclin E-Cdk2 accelerates entry into S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. We identified 20 amino acids in cyclin E as a centrosomal localization signal (CLS) essential for both centrosomal targeting and promoting DNA synthesis. Expressed wild-type, but not mutant, CLS peptides localized on the centrosome, prevented endogenous cyclin E and cyclin A from localizing to the centrosome, and inhibited DNA synthesis. Ectopic cyclin E localized to the centrosome and accelerated S phase entry even with mutations that abolish Cdk2 binding, but not with a mutation in the CLS. These results suggest that cyclin E has a modular centrosomal-targeting domain essential for promoting S phase entry in a Cdk2-independent manner.

Divertor plasma characteristics in the Large Helical Device (LHD) have been investigated mainly by using Langmuir probes. The three-dimensional structure of the helical divertor, which is naturally produced in the heliotron-type magnetic configuration, is clearly seen in the measured particle and power deposition profiles on the divertor plates. These observations are consistent with the numerical results of field line tracing. The particle flux to the divertor plates increases almost linearly with the line averaged density. The high-recycling regime and divertor detachment, which are observed in tokamaks, have not been observed even during high density discharges with low input power. Both electron density and temperature decrease with increasing radius in the stochastic layer with open field lines, and at the divertor plate they become fairly low compared with those at the last closed flux surface. This means the reduction of pressure along the magnetic field lines occurs in the open field line region in LHD.

Pulmonary lymphangioleiomyomatosis (LAM), a disease of young women, is characterized by proliferation of immature-appearing smooth-muscle cells (LAM cells) in the lungs and abdomen. LAM cells react with monoclonal antibody HMB45, which recognizes a 100-kD glycoprotein (gp100) originally found in human melanoma cells. We investigated the expression and the subcellular localization of gp100 in lung tissue from patients with LAM and in human melanoma cell lines (Malme-3M, A2058, and CHL-1), and the relationship between this expression and cellular proliferation. Binding sites for HMB45 antibody in melanoma and LAM cells were located in cytoplasmic granules resembling immature melanosomes. LAM cells reactive for proliferating-cell nuclear antigen (PCNA), a marker of cellular proliferation, were spindle-shaped, in contrast to the large, epithelioid cells reacting with HMB45 antibody. In accord with this finding, we observed an inverse relationship between the immunostaining for HMB45 antibody and PCNA in LAM and melanoma cells. Thus, LAM and melanoma cells are heterogeneous with respect to their stages of proliferation and their expression of melanoma antigens. PCNA-positive cells, which are more likely to be negative for reactivity with HMB45 antibody, may be more relevant to the progression of LAM than are HMB45-positive cells, which are the hallmark of LAM.

Aberrant centrosome duplication is observed in many tumor cells and may contribute to genomic instability through the formation of multipolar mitotic spindles. Cyclin-dependent kinase 2 (Cdk2) is required for multiple rounds of centrosome duplication in Xenopus egg extracts but not for the initial round of replication. Egg extracts undergo periodic oscillations in the level of free calcium. We show here that chelation of calcium in egg extracts or specific inactivation of calcium/calmodulin-dependent protein kinase II (CaMKII) blocks even initial centrosome duplication, whereas inactivation of Cdk2 does not. Duplication can be restored to inhibited extracts by addition of CaMKII and calmodulin. These results indicate that calcium, calmodulin, and CaMKII are required for an essential step in initiation of centrosome duplication. Our data suggest that calcium oscillations in the cell cycle may be linked to centrosome duplication.

Circular RNA replicons have been reported in plants and, in one case, in animal cells. We describe such an element in yeast. In certain yeast strains, a 20S RNA species appears on transfer of cells to acetate medium. This phenotype shows cytoplasmic (non-Mendelian) inheritance and the 20S RNA is associated with 23-kDa protein subunits as a 32S particle. We demonstrate that yeast 20S RNA is an independent replicon with no homology to host genomic, mitochondrial, or 2-pm plasmid DNA or to the L-A, L-BC, or Ml doublestranded RNA viruses of yeast. The circularity of the 20S RNA is shown by the apparent absence of 3' and 5' ends, by two-dimensional gel electrophoresis, and by electron microscopy. Replication of yeast 20S RNA proceeds through an RNA-RNA pathway, and a 10,000-fold amplification occurs on shift to acetate medium. The copy number of 20S RNA is also reduced severalfold by the SKI gene products, a host antiviral system that also lowers the copy numbers of yeast doublestranded RNA viruses. Yeast 20S RNA and the hepatitis 8 virus show some similarities.Among circular single-stranded RNA replicons, the viroids are 300-to 400-base infectious agents of plants that lack a coat and encode no proteins. Viroid-like satellite RNAs resemble viroids but are encapsidated in a coat provided by an RNA plant virus (1-4). The hepatitis 8 virus RNA (1700 bases) is also a circular single-stranded RNA and encodes at least one protein, the 8 antigen, which is necessary for its replication and with which it is associated in cells or in viral particles supplied by hepatitis B virus (5-10). Many of these circular RNA replicons have been found to carry out self-cleavage of monomer genome segments from the multimers generated in the replication process and self-ligation of monomers to form circles. We have now found a single-stranded circular RNA replicon in Saccharomyces cerevisiae.In addition to the known double-stranded RNA viruses (11) and retroviruses (12) of yeast, there are a number of cytoplasmic genetic elements whose basis is less well understood. A 20S RNA species in yeast was found to be amplified in potassium acetate medium, the same medium used to induce meiosis and sporulation (13,14). Garvik and Haber (15) showed that ability to amplify this RNA was independent of sporulation competence and was cytoplasmically inherited, but whether this 20S RNA itself or only the ability to amplify it was cytoplasmically inherited was unclear. Wejksnora andHaber (16) Preparation of Yeast 20S RNA. Cells were grown to logarithmic phase in YPAD medium, shifted into 1% potassium acetate (16), and harvested, and protoplasts were prepared by incubation with zymolyase (Kirin Brewery, Japan) at 0.5 mg/ml (17). Protoplasts were disrupted in 0.2% SDS/0.3 M NaCl/0.1 M EDTA/2 mM dithiothreitol with bentonite (Sigma) at 1 mg/ml. The sample was extracted four times with phenol/chloroform and nucleic acids were precipitated with 2 volumes of ethanol. The 20S RNA was enriched from this total RNA preparation (from 6 g of cells) by isopy...

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As the finalization of the hydrogen experiment towards the deuterium phase, the exploration of the best performance of the hydrogen plasma was intensively performed in the Large Helical Device (LHD). High ion and electron temperatures, Ti, Te, of more than 6 keV were simultaneously achieved by superimposing the high power electron cyclotron resonance heating (ECH) on the neutral beam injection (NBI) heated plasma. Although flattening of the ion temperature profile in the core region was observed during the discharges, one could avoid the degradation by increasing the electron density. Another key parameter to present plasma performance is an averaged beta value . The high regime around 4 % was extended to an order of magnitude lower than the earlier collisional regime. Impurity behaviour in hydrogen discharges with NBI heating was also classified with the wide range of edge plasma parameters. Existence of no impurity accumulation regime where the high performance plasma is maintained with high power heating > 10 MW was identified. Wide parameter scan experiments suggest that the toroidal rotation and the turbulence are the candidates for expelling impurities from the core region.