The highly conserved RCN family of proteins regulates the serine/ threonine protein phosphatase calcineurin, which is required for the expression of genes involved in Ca 2؉ -dependent processes, such as the control of memory, apoptosis, T cell activation, cell cycle, Ca 2؉ -homeostasis, and skeletal and cardiac muscle growth and differentiation. However, RCNs regulate calcineurin through two paradoxical actions: they act as feedback inhibitors of calcineurin, whereas their phosphorylation stimulates calcineurin. Here we show that phosphorylation of yeast RCN, Rcn1, triggers degradation through the SCF Cdc4 ubiquitin ligase complex. Degradation of phosphorylated Rcn1 is required to mitigate inhibition of calcineurin by Rcn1 and results in activation of calcineurin activity in response to Ca 2؉ as well as in reactivation of calcineurin in response to changes in Ca 2؉ concentration. The SCF Cdc4 -dependent degradation required phosphorylation of Rcn1 by Mck1, a member of the GSK3 family of protein kinases, and was promoted by Ca 2؉ . However, such degradation was counteracted by dephosphorylation of Rcn1, which was promoted by Ca 2؉ -stimulated calcineurin. Thus, calcineurin activity is fine-tuned to Ca 2؉ signals by mechanisms that have opposite functions. Our results identify the molecular mechanism of Rcn1 phosphorylation-induced stimulation of the phosphatase activity of calcineurin. The results provide insight into the mechanism involved in maintaining proper responses to Ca 2؉ signals.calcium signaling ͉ DSCR1/MCIP ͉ feedback inhibitor ͉ proteolysis

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Both biological treatment processes including conventional activated sludge (CAS) and biological nutrient removal (BNR) processes, and physico-chemical treatment processes including ozonation process and Title 22 process consisting of coagulation, sedimentation and filtration followed by UV or chlorination disinfection after the above biological processes, were compared from the viewpoint of removal efficiency. 66 pharmaceuticals including antibiotics, analgesics, psychoneurotic agents were measured with SPE-LC/MS/MS. 26 compounds out of 66 were detected in the influent ranging ng/L to microg/L order. Particularly, disopyramide, sulpiride, and dipyridamole that have been rarely detected before in the WWTP, occurred at concentration levels of more than 100 ng/L. The total concentration of the individual pharmaceuticals in the influent was efficiently removed by 80% during the biological treatment. But removal efficiencies of carbamazepine and crotamiton were less than 30%. The total concentration of the individual pharmaceuticals in the effluent from CAS process was 1.5 times higher than that from BNR process. Further, the total concentration of the individual pharmaceuticals in the discharge from WWTPs applying ozonation following activated sludge process was reduced to less than 20%. Physico-chemical treatment train called Title 22 treatment after CAS could not efficiently remove the pharmaceuticals. However, ozonation process followed by biological activated carbon process could efficiently reduce all the residual pharmaceuticals below their quantification limits.

Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl þ leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl þ stem cells is needed to cure leukemias caused by Bcr-Abl þ cells. Human Bcr-Abl þ cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl þ sublines by selection in long-term hypoxic cultures (1.0% O 2 ). Interestingly, HA-Bcr-Abl þ cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher b-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl þ cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl þ cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl þ leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl þ leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment.

IntroductionPhiladelphia (Ph) chromosome results from a reciprocal translocation between chromosomes 9 and 22 and generates the BCR-ABL chimera protein, the cause of chronic myeloid leukemia (CML) and Ph ϩ acute lymphoid leukemia (ALL). The ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, has dramatically changed the first-line therapy of CML. 1 Most patients with newly diagnosed CML with chronic phase, when treated with imatinib, achieve durable responses. However, emergence of refractory disease and relapse have frequently been reported, particularly in patients with CML with advanced-stage disease and patients with Ph ϩ ALL. 2,3 Among several mechanisms of resistance, point mutations within the ABL kinase domain that interfere with imatinib binding are the most critical cause of imatinib resistance. 4,5 To overcome these imatinib resistance mechanisms, 4 secondgeneration ABL TKIs have been developed: dasatinib, 6 nilotinib, 7 bosutinib 8 and bafetinib (formerly INNO-406). 9,10 Despite promising clinical results from these second-generation ABL TKIs for most patients with imatinib-resistant BCR-ABL ϩ leukemia, the mutation of threonine 315 to isoleucine (T315I) confers resistance to all these TKIs. 11,12 Thus, identification of novel agents for the effective treatment of patients with CML with T315I is an important and challenging task. 13 Aurora kinases A and B are a family of serine/threonine kinases involved in many cellular functions. [14][15][16] Inappropriate expression of these enzymes in certain cancers may result in aneuploidy and carcinogenesis. 17 Consequently, the potential therapeutic value of targeting Aurora kinases has become a focus of anticancer therapy. 14 Recently, we identified an Aurora kinase inhibitor, AT9283 (1-cyclopropyl-3[5-morpholin-4yl methyl-1H-benzomidazol-2-yl]-urea) by way of structure-based optimization of a ligand-efficient pyrazole-benzimidazole fragment. X-ray crystallographic structures were generated with the use of a novel soakable form of Aurora A and were used to drive the optimization toward potent (half-maximal inhibition constant [IC 50 ] Ͻ 3nM) dual Aurora A/B inhibitor. AT9283 that also potently inhibits several kinases, including Janus kinase-2 (JAK2) and JAK3 (1.2 and 1.1nM, respectively), c-ABL (110nM), and ABL/T315I (4nM), is currently under evaluation in phase 1 clinical trials for metastatic solid tumors and hematologic malignancies. 18,19 Here, we report the putative mechanism by which AT9283 binds to BCR-ABL/T315I and its activity against imatinib-resistant BCR-ABL ϩ leukemic cells, including those with the T315I mutation. Methods Reagent and cell linesAT9283 was synthesized by Astex Therapeutics Ltd ( Figure 1A). 18 Human CML cell lines (K562, MEG-01, BV173, KU812, MYL, KT-1, and The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC sectio...