143 research outputs found

    Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species

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    CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis

    Activity Assay of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Triple-Negative Breast Cancer Cells Using Peptide-Conjugated Magnetic Beads

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    Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with limited treatment options. Epidermal growth factor receptor I (EGFR) has emerged as a promising target in TNBC. Limited success of the EGFR kinase inhibiting small molecules in clinical trials may be attributed in part to inaccuracy in identifying EGFR signatures in patient tumors. In light of the absence of a simple correlation between EGFR expression and its degree of activation, a simple and reliable tool that can quantify EGFR kinase activity in tumor samples may be of therapeutic value in predicting patient-specific EGFR targeted therapies. This study reports the development of an assay that can quantitatively profile EGFR kinase activities and inhibitor sensitivities in TNBC cell lysates by using peptide reporters covalently tethered to magnetic beads in a controlled orientation. The use of magnetic beads provides rapid sample handling and easy product isolation. The potential of this approach was demonstrated by screening a set of five clinically relevant EGFR tyrosine kinase inhibitors. Formatted for microwell plates, this magnetic bead-based kinase assay may be used as a complementary approach for direct high-throughput screening of small molecule inhibitors.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140099/1/adt.2012.454.pd

    CDK-dependent Hsp70 phosphorylation controls G1 cyclin abundance and cell-cycle progression

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    In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

    CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

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    In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

    Dynamic Critical Behavior of an Extended Reptation Dynamics for Self-Avoiding Walks

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    We consider lattice self-avoiding walks and discuss the dynamic critical behavior of two dynamics that use local and bilocal moves and generalize the usual reptation dynamics. We determine the integrated and exponential autocorrelation times for several observables, perform a dynamic finite-size scaling study of the autocorrelation functions, and compute the associated dynamic critical exponents zz. For the variables that describe the size of the walks, in the absence of interactions we find z≈2.2z \approx 2.2 in two dimensions and z≈2.1z\approx 2.1 in three dimensions. At the θ\theta-point in two dimensions we have z≈2.3z\approx 2.3.Comment: laTeX2e, 32 pages, 11 eps figure

    UV-Optical Pixel Maps of Face-On Spiral Galaxies -- Clues for Dynamics and Star Formation Histories

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    UV and optical images of the face-on spiral galaxies NGC 6753 and NGC 6782 reveal regions of strong on-going star formation that are associated with structures traced by the old stellar populations. We make NUV--(NUV-I) pixel color-magnitude diagrams (pCMDs) that reveal plumes of pixels with strongly varying NUV surface brightness and nearly constant I surface brightness. The plumes correspond to sharply bounded radial ranges, with (NUV-I) at a given NUV surface brightness being bluer at larger radii. The plumes are parallel to the reddening vector and simple model mixtures of young and old populations, thus neither reddening nor the fraction of the young population can produce the observed separation between the plumes. The images, radial surface-brightness, and color plots indicate that the separate plumes are caused by sharp declines in the surface densities of the old populations at radii corresponding to disk resonances. The maximum surface brightness of the NUV light remains nearly constant with radius, while the maximum I surface brightness declines sharply with radius. An MUV image of NGC 6782 shows emission from the nuclear ring. The distribution of points in an (MUV-NUV) vs. (NUV-I) pixel color-color diagram is broadly consistent with the simple mixture model, but shows a residual trend that the bluest pixels in (MUV-NUV) are the reddest pixels in (NUV-I). This may be due to a combination of red continuum from late-type supergiants and [SIII] emission lines associated with HII regions in active star-forming regions. We have shown that pixel mapping is a powerful tool for studying the distribution and strength of on-going star formation in galaxies. Deep, multi-color imaging can extend this to studies of extinction, and the ages and metallicities of composite stellar populations in nearby galaxies.Comment: LaTeX with AASTeX style file, 29 pages with 12 figures (some color, some multi-part). Accepted for publication in The Astrophysical Journa

    The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair

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    P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments
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