Nutrient limitations alter cell division control and chromosome segregation through growth-related kinases and phosphatases

In dividing fission yeast Schizosaccharomyces pombe cells, the balance between Wee1 kinase and Cdc25 phosphatase which control the cyclin-dependent kinase (CDK) at the G2–M transition determines the rod-shaped cell length. Under nitrogen source starvation or glucose limitation, however, cell size determination is considerably modulated, and cell size shortening occurs for wild-type cells. For several mutants of kinases or phosphatases, including CDK, target of rapamycin complex (TORC) 1 and 2, stress-responsive mitogen-activated protein kinase (MAPK) Sty1/Spc1, MAPK kinase Wis1, calcium- and calmodulin-dependent protein kinase kinase-like Ssp1, and type 2A and 2A-related phosphatases inhibitor Sds23, this cell shortening does not normally occur. In tor1 and ssp1 mutants, cell elongation is observed. Sds23 that binds to and inhibits 2A and 2A-related phosphatases is synergistic with Ssp1 in the cell size determination and survival under low glucose and nitrogen source. Tor2 (TORC1) is required for growth, whereas Tor1 (TORC2) is needed for determining division size according to different nutrient conditions. Surprisingly, in growth-diminished tor2 mutant or rapamycin-treated cells, the requirement of separase/Cut1-securin/Cut2 essential for chromosome segregation is greatly alleviated. By contrast, defects of tor1 with secruin/cut2 or overproduction of Cut1 are additive. While Tor1 and Tor2 are opposite in their apparent functions, both may actually coordinate cell division with growth in response to the changes in nutrients

A Method for Analyzing the Ubiquitination and Degradation of Aurora-A

The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium

Selective Catalytic Reduction of Nitric-oxide With Ammonia Using Moo3/tio2 - Catalyst Structure and Activity

A series of titania supported MoO3 catalysts (0-20 wt.-% MoO3) were prepared by dry impregnation. The influence of the MoO3 content on their catalytic performance for the selective catalytic reduction (SCR) of nitric oxide by ammonia in the presence of oxygen, as well as on their textural and structural properties has been studied. The samples were characterized by XRD, XPS, IR, and BET and porosimetry measurements. The coverage of the TiO2 support by surface polymeric molybdenum species (where molybdenum is octahedrally coordinated) increases with the molybdenum loading. The formation of a layer of these interacting species on top of the titania surface is complete in the range 15-20 wt.-% MoO3. The formation of crystallites of bulk MoO3 starts before the completion of this surface layer (at around 10 wt.-% MoO3) and increases progressively as the molybdenum loading increases from 10 to 20 wt.-% MoO3. The SCR activity of the MoO3/TiO2 catalysts increases as the MoO3 content increases to 15 wt.-% and then, for a further increase of the molybdenum loading, it slightly decreases. No specific influence of the molybdenum content on the resistance of catalysts towards SO2 was observed; the same slight deactivation took place, when the SCR activity was measured in the presence of SO2 in the feed, for all samples. Our results indicate that the octahedrally coordinated polymeric molybdenum surface species are mainly responsible for the exhibited SCR activity of the MoO3/TiO2 catalysts

Thermal oxidation under oxygen of zirconium nitride studied by XPS, DRIFTS, TG-MS

The studies of oxidation of nitrides or oxynitrides compounds are important since their electrical, thermal, catalytical and reactional properties are drastically modified by the oxidation layer encountered on top of these ceramics. In this paper we present the thermal oxidation of ZrN under O-2, in order to have a better understanding of the intermediate species encountered during the reaction, in the frame of low temperature nitridation of ceramics. The analysis of gas reaction products during oxidation and the confrontation of XPS and DRIFTS results on partially oxidized samples allow to evidence the formation of a stable Zr-N-N-O-Zr intermediate. (C) 1998 Elsevier Science Limited. All rights reserved

A Way To Inhibit So2 Poisoning of Scr Catalysts By Fine-tuning of the Composition and the Preparation Method of the Support

Two series of mixed SiO2-TiO2 and Al2O3-TiO2 supports were prepared by the following methods : precipitation from TiCl4 solution with aqueous ammonia, impregnation with titanium isopropoxide solution in isopropanol, and grafting by reaction of TiCl4 with the hydroxyl groups of the main carrier (SiO2 or Al2O3). The vanadia catalysts, resulting from the impregnation of these mixed supports with an aqueous solution of vanadium oxalate, were tested in the selective catalytic reduction (SCR) of NO with NH3 in the presence of O2. BET, XRD and XPS were used to characterise the samples. All these catalysts were less active than a standard TiO2-V2O5 catalyst when tested with a SO2-free feed. However, in the presence of 4000ppm SO2 in the feed they were more resistant and more active than the TiO2-V2O5 catalyst. Our results indicate that the dispersion of TiO2 on the main carrier is a key factor for preparing a performant SCR catalyst. The grafting and impregnation methods for the Al2O3-TiO2 and the impregnation for the SiO2-TiO2 supports were found to be the most effective preparation techniques for dispersing TiO2, and for giving the most performant and resistant, towards SQ2 poisoning, catalysts

The CHK2-BRCA1 tumour suppressor pathway ensures chromosomal stability in human somatic cells

Chromosomal instability (CIN) is a major hallmark of human cancer and might contribute to tumorigenesis. Genes required for the normal progression of mitosis represent potential CIN genes and, as such, are important tumour suppressors. The Chk2 kinase and its downstream targets p53 and Brca1 are tumour suppressors that have been functionally linked to the DNA damage response pathway. Here, we report a function of Chk2, independent of p53 and DNA damage, that is required for proper progression of mitosis, and for the maintenance of chromosomal stability in human somatic cells. Depletion of Chk2 or abrogation of its kinase activity causes abnormal mitotic spindle assembly associated with a delay in mitosis, which promotes the generation of lagging chromosomes, chromosome missegregation and CIN, while still allowing survival and growth. Furthermore, we have identified Brca1 as a mitotic target of the Chk2 kinase in the absence of DNA damage. Accordingly, loss of BRCA1 or its Chk2-mediated phosphorylation leads to spindle formation defects and CIN. Thus, the CHK2-BRCA1 tumour suppressor pathway is required for chromosomal stability, which might contribute to their tumour suppressor function

Altering microtubule dynamics is synergistically toxic with spindle assembly checkpoint inhibition.

Chromosomal instability (CIN) and aneuploidy are hallmarks of cancer. As most cancers are aneuploid, targeting aneuploidy or CIN may be an effective way to target a broad spectrum of cancers. Here, we perform two small molecule compound screens to identify drugs that selectively target cells that are aneuploid or exhibit a CIN phenotype. We find that aneuploid cells are much more sensitive to the energy metabolism regulating drug ZLN005 than their euploid counterparts. Furthermore, cells with an ongoing CIN phenotype, induced by spindle assembly checkpoint (SAC) alleviation, are significantly more sensitive to the Src kinase inhibitor SKI606. We show that inhibiting Src kinase increases microtubule polymerization rates and, more generally, that deregulating microtubule polymerization rates is particularly toxic to cells with a defective SAC. Our findings, therefore, suggest that tumors with a dysfunctional SAC are particularly sensitive to microtubule poisons and, vice versa, that compounds alleviating the SAC provide a powerful means to treat tumors with deregulated microtubule dynamics