cdc2 phosphorylation is required for its interaction with cyclin.

Activation of the cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins. We show that in vitro translated human cdc2 binds very poorly to A and B cyclins, unless it has been preincubated with a Xenopus egg extract. This results in the phosphorylation of cdc2 which allows binding to cyclins. The replacement of Thr161, a residue conserved and phosphorylated in other protein kinases, with valine inhibits cdc2 association with A and B cyclins. In addition, mutations in the amino-terminus of cdc2 and within the conserved 'PSTAIR' region strongly inhibit binding. The Thr161Val mutation causes a lethal phenotype in the fission yeast Schizosaccharomyces pombe, while replacement of Thr161 with glutamic acid, potentially mimicking phosphorylation, causes uncoordination of mitosis and multiple cytokinesis. These results suggest that a threonine phosphorylation/dephosphorylation cycle is involved in regulating cdc2 function.     

Juvenile hormone-like substances can induce vitellogenesis in the tick Ornithodoros moubata (Acarina: Argasidae)

Topical application of different juvenile hormone analogs (JHA) or of a mixture of stereoisomers of insect juvenile hormone (JH) 1 and 3 to fed virgin female Ornithodoros moubata immediately after feeding induced vitellogenesis and egg-laying in up to 70% of treated females. In controls only 13.7% oviposited. The eggs were sterile, with abnormal shape, but their number versus the weight of engorged females was normal or sometimes greater than in mated females. However, preoviposition period was longer than in mated females.

It was more difficult to induce egg-laying by similar topical applications 100 days after feeding of virgin females. A maximum of 58% of ovipositing females was obtained with a very high dosage of JH mixture (500 fig). Injection of this mixture into the females was more potent; 15 to 50 fig induced oviposition in about 60% of the females. The preoviposition period was also longer than in control females.

Our results suggest the presence of a JH-like substance which is involved in the hormonal control of vitellogenesis. However, since natural isomers of JH were much less efficient than isomeric mixtures or JHA, we suppose that the natural tick hormone does not correspond to JH, but rather to a JH-like substance.     

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.     

Monitoring of temperature effects on animal cell metabolism in a packed bed process

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the data were used for process development. At 37 degrees C cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate production rates increased continually. Reduction of the temperature to 33.5 degrees C resulted in a lowering of these metabolic rates while having no effect on cell proliferation. Subsequent reduction of the temperature to 32 degrees C resulted in stabilization of the cell concentration at a high density (3.6 x 10(7) cell per mL of packed bed). In addition, the specific production rate of the protein of interest increased by a factor of 6 compared to the value at 37 degrees C. During the stationary phase at 32 degrees C, all other specific metabolic rates could be controlled to low and constant levels.     

Human CDC25B and CDC25C differ by their ability to restore a functional checkpoint response after gene replacement in fission yeast

In fission yeast, inactivation of the Cdc25 phosphatase by checkpoint kinases participates in the signaling cascade that temporarily stops cell cycle progression after DNA damage. In human, CDC25B and C are also known to be targeted by a similar checkpoint machinery. We have examined by homologous recombination, whether CDC25B and CDC25C were able to substitute for the function of fission yeast Cdc25. We demonstrate that (i) CDC25B and C efficiently replace Cdc25 for vegetative growth, (ii) CDC25C is able to restore a functional checkpoint in response to ionizing radiation in both a Chk1- and Cds1-dependent manner, (iii) CDC25B and C are equally efficient in the response to UV irradiation, CDC25B being only dependent on Chk1, while CDC25C depends on both Chk1 and Cds1, and (iv) CDC25C is able to restore a functional DNA replication checkpoint induced by hydroxyurea in a Cds1-dependent manner. The consequences of these findings on our current view of the checkpoint cascade are discussed.     

PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation

Regulation of the intracellular localisation of its actors is one of the key mechanisms underlying cell cycle control. CDC25 phosphatases are activators of Cyclin-Dependent Kinases (CDK) that undergo nucleo-cytoplasmic shuttling during the cell cycle and in response to checkpoint activation. Here we report that the protein kinase PKB/Akt phosphorylates CDC25B on serine 353, resulting in a nuclear export-dependent cytoplasmic accumulation of the phosphatase. Oxidative stress activates PKB/Akt and reproduces the effect on CDC25B phosphorylation and localisation. However, inhibition of PKB/Akt activity only partially reverted the effect of oxidative stress on CDC25B localisation and mutation of serine 353 abolishes phosphorylation but only delays nuclear exclusion. These results indicate that additional mechanisms are also involved in preventing nuclear import of CDC25B. Our findings identify CDC25B as a target of PKB/Akt and provide new insight into the regulation of its localisation in response to stress-activated signalling pathways.     

Characterization of the antiproliferative activity of Xylopia aethiopica

Background

Xylopia aethiopica, a plant found throughout West Africa, has both nutritional and medicinal uses. The present study aims to characterize the effects of extracts of this plant on cancer cells.

Results

We report that X. aethiopica extract prepared with 70% ethanol has antiproliferative activity against a panel of cancer cell lines. The IC50 was estimated at 12 ??g/ml against HCT116 colon cancer cells, 7.5 ??g/ml and > 25 ??g/ml against U937 and KG1a leukemia cells, respectively. Upon fractionation of the extract by HPLC, the active fraction induced DNA damage, cell cycle arrest in G1 phase and apoptotic cell death. By using NMR and mass spectrometry, we determined the structure of the active natural product in the HPLC fraction as ent-15-oxokaur-16-en-19-oic acid.

Conclusion

The main cytotoxic and DNA-damaging compound in ethanolic extracts of Xylopia aethiopica is ent-15-oxokaur-16-en-19-oic acid.     

Etoposide and adriamycin but not genistein can activate the checkpoint kinase Chk2 independently of ATM/ATR.

We have investigated the effects of three unrelated topoisomerase 2 inhibitors, genistein, adriamycin, and etoposide, on phosphorylation/activation of the checkpoint kinase Chk2 in normal or ATM-deficient (ATM-) human fibroblasts and in cells overexpressing a catalytically inactive ATR kinase. We demonstrate that genistein activates Chk2 in a strictly ATM-dependent manner, whereas etoposide and adriamycin can trigger Chk2 activation in long-term cultures of ATM- cells. Moreover, these two latter genotoxic compounds were found to activate Chk2 in fibroblasts expressing the dominant negative form of ATR. We also report a significant decrease in the accumulation in G2-phase of ATM- cells when genistein did not activate Chk2. In conclusion, our results strongly support that activation of Chk2 could be dependent on the type and/or extent of DNA damage and under the control of either an ATM-dependent or an ATM and, maybe, an ATR-independent pathway.