Rolofylline, an adenosine A1−receptor antagonist, in acute heart failure

Background: Worsening renal function, which is associated with adverse outcomes, often develops in patients with acute heart failure. Experimental and clinical studies suggest that counterregulatory responses mediated by adenosine may be involved. We tested the hypothesis that the use of rolofylline, an adenosine A1−receptor antagonist, would improve dyspnea, reduce the risk of worsening renal function, and lead to a more favorable clinical course in patients with acute heart failure. Methods: We conducted a multicenter, double-blind, placebo-controlled trial involving patients hospitalized for acute heart failure with impaired renal function. Within 24 hours after presentation, 2033 patients were randomly assigned, in a 2:1 ratio, to receive daily intravenous rolofylline (30 mg) or placebo for up to 3 days. The primary end point was treatment success, treatment failure, or no change in the patient’s clinical condition; this end point was defined according to survival, heart-failure status, and changes in renal function. Secondary end points were the post-treatment development of persistent renal impairment and the 60-day rate of death or readmission for cardiovascular or renal causes. Results: Rolofylline, as compared with placebo, did not provide a benefit with respect to the primary end point (odds ratio, 0.92; 95% confidence interval, 0.78 to 1.09; P=0.35). Persistent renal impairment developed in 15.0% of patients in the rolofylline group and in 13.7% of patients in the placebo group (P=0.44). By 60 days, death or readmission for cardiovascular or renal causes had occurred in similar proportions of patients assigned to rolofylline and placebo (30.7% and 31.9%, respectively; P=0.86). Adverse-event rates were similar overall; however, only patients in the rolofylline group had seizures, a known potential adverse effect of A1-receptor antagonists. Conclusions: Rolofylline did not have a favorable effect with respect to the primary clinical composite end point, nor did it improve renal function or 60-day outcomes. It does not show promise in the treatment of acute heart failure with renal dysfunction. (Funded by NovaCardia, a subsidiary of Merck; ClinicalTrials.gov numbers, NCT00328692 and NCT00354458.

Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway

LEM-3 – A LEM Domain Containing Nuclease Involved in the DNA Damage Response in C. elegans

The small nematode Caenorhabditis elegans displays a spectrum of DNA damage responses similar to humans. In order to identify new DNA damage response genes, we isolated in a forward genetic screen 14 new mutations conferring hypersensitivity to ionizing radiation. We present here our characterization of lem-3, one of the genes identified in this screen. LEM-3 contains a LEM domain and a GIY nuclease domain. We confirm that LEM-3 has DNase activity in vitro. lem-3(lf) mutants are hypersensitive to various types of DNA damage, including ionizing radiation, UV-C light and crosslinking agents. Embryos from irradiated lem-3 hermaphrodites displayed severe defects during cell division, including chromosome mis-segregation and anaphase bridges. The mitotic defects observed in irradiated lem-3 mutant embryos are similar to those found in baf-1 (barrier-to-autointegration factor) mutants. The baf-1 gene codes for an essential and highly conserved protein known to interact with the other two C. elegans LEM domain proteins, LEM-2 and EMR-1. We show that baf-1, lem-2, and emr-1 mutants are also hypersensitive to DNA damage and that loss of lem-3 sensitizes baf-1 mutants even in the absence of DNA damage. Our data suggest that BAF-1, together with the LEM domain proteins, plays an important role following DNA damage – possibly by promoting the reorganization of damaged chromatin

Common variation in PHACTR1 is associated with susceptibility to cervical artery dissection

Cervical artery dissection (CeAD), a mural hematoma in a carotid or vertebral artery, is a major cause of ischemic stroke in young adults although relatively uncommon in the general population (incidence of 2.6/100,000 per year). Minor cervical traumas, infection, migraine and hypertension are putative risk factors, and inverse associations with obesity and hypercholesterolemia are described. No confirmed genetic susceptibility factors have been identified using candidate gene approaches. We performed genome-wide association studies (GWAS) in 1,393 CeAD cases and 14,416 controls. The rs9349379[G] allele (PHACTR1) was associated with lower CeAD risk (odds ratio (OR) = 0.75, 95% confidence interval (CI) = 0.69-0.82; P = 4.46 × 10(-10)), with confirmation in independent follow-up samples (659 CeAD cases and 2,648 controls; P = 3.91 × 10(-3); combined P = 1.00 × 10(-11)). The rs9349379[G] allele was previously shown to be associated with lower risk of migraine and increased risk of myocardial infarction. Deciphering the mechanisms underlying this pleiotropy might provide important information on the biological underpinnings of these disabling conditions

The Presence of SA2–12xA on Chromosome Arms Correlates with Cohesion between Sister Chromatid Arms

<div><p>(A) Cells were cultured in the absence or presence of different amounts of doxycycline as indicated. After arrest in nocodazole for 3 h, cells were fixed, spread on glass slides, and stained with Giemsa (photomicrographs, above). Single chromosomes (indicated by a box) are shown at higher magnification in the lower right corners. The number of cells with chromosome arms that had opened (arms open) or that were connected (arms closed) was scored as indicated (bar graphs, below). Scale bar 10 μm.</p> <p>(B) Whole-cell extracts were prepared from HeLa cells expressing SA2–12xA-myc after treatment with increasing amounts of doxycycline (0, 0.2, and 2.0 μg/ml). The ratio of exogenous SA2–12xA-myc to endogenous SA2 was visualized by immunoblotting with antibodies to SA2. The position of molecular weight markers is indicated on the right.</p></div

Identification of Mitosis-Specific Phosphorylation Sites on Human Cohesin

<div><p>(A and B) Cohesin was immunoprecipitated by antibody 447 (which recognizes SA1 and SA2) from extracts prepared from HeLa cells that were either arrested in S-phase by hydroxyurea (HU) or in mitosis by nocodazole (Noc). Cohesin was eluted by buffer of low pH and analyzed by (A) silver staining and (B) immunoblotting with antibodies to cohesin subunits and phosphorylated threonine (P-Thr).</p> <p>(C) Schematic representation of the phosphorylation sites on Scc1 and SA2 that were identified by mass spectrometry, and of the mutant versions of the proteins that have been generated. The star indicates a phosphorylation site that was found in both interphase and mitotic Scc1. All SA2 constructs used for in vitro experiments lack the 69 N-terminal amino acids. SA2-WT-myc and SA2–12xA-myc cell lines contain the entire open reading frame of 1,231 amino acids.</p></div

<i>lem-3(op444)</i> phenotypes following DNA damage.

<p>Wild-type animals and <i>lem-3(op444)</i> mutants were treated 24 h post L4/adult molt with (A) X-rays (B) UV-C, or (C) as L4 larvae with cisplatin. F1 embryonic lethality was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024555#s4" target="_blank">Materials and Methods</a>. Data shown represent the average of three independent experiments ± S.D. The progeny of 10 worms (A, B) or 5 worms (C) were analysed for each experiment. (D) Late radiation phenotypes. L1 animals were irradiated with 60 Gy. The uncoordinated phenotype (Unc) and protruding vulva phenotype (P-vul) were scored only for those animals that reached adulthood (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024555#s4" target="_blank">Materials and Methods</a>). Data shown represent the average of three independent experiments ± S.D. (n for wild type, <i>lem-3(op444)</i> and <i>cku-80(ok861)</i> are 305, 236 and 373 respectively).</p

Plk1 Facilitates Cleavage of Human Scc1 by Separase In Vitro

<div><p>(A) Recombinant, ³⁵S-labeled, wild-type and mutant Scc1 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030069#pbio-0030069-g001" target="_blank">Figure 1</a>C) tagged with 9xmyc at the C terminus were incubated with human separase. Recombinant human GST-Plk1 was added to the reaction mixtures where indicated. Samples were withdrawn from the reactions at the indicated time points and analyzed by SDS-PAGE followed by immunoblotting (anti-myc) and Phosphorimager analysis (³⁵S exposure). Arrows indicate full length Scc1-myc (fl), C- and N-terminal fragments resulting from cleavage at Arg¹⁷² (Ct #1, Nt #1, respectively), and C- and N-terminal fragments resulting from cleavage at Arg⁴⁵⁰ (Ct #2, Nt #2, respectively). The lower parts of the membrane or gels were exposed longer than the upper parts. The enhancement of cleavage at Arg¹⁷² by Plk1 can be seen particularly well by comparing the intensities of the N-terminal fragments (Nt #1). Note that in the autoradiographs a band can be seen (particularly clearly in the lanes representing the zero time points) that has almost the same electrophoretic mobility as cleavage product Ct #1. This band is distinct from Ct #1 because it migrates a slightly shorter distance and because it is also present in the absence of separase. This band was therefore not included in the quantification in (B).</p> <p>(B) Quantification of the abundance of Scc1-myc and the Scc1-myc cleavage fragments in the assay shown in the left autoradiograph of (A). For the quantification, autoradiographs of identical exposure were used. The sum of the intensities of full-length and all cleavage fragments was set to 100%. Signal intensities for N- and C-terminal fragments resulting from cleavage at the same site were summed.</p> <p>(C) Chromatin fractions were prepared from HeLa cells stably expressing either wild-type Scc1-myc or the mutant Scc1-S⁴⁵⁴A-myc, and were incubated in either interphase or mitotic <i>Xenopus</i> egg extract. Mitosis-specific cleavage of Scc1 was detected by immunoblotting with myc antibodies.</p></div

Phosphorylation of SA2 Is Required for Efficient Resolution of Sister Chromatid Arms during Prometaphase and Metaphase

<div><p>(A) HeLa cells expressing SA2-WT-myc or SA2–12xA-myc were spread on glass slides and chromosomes were stained with Giemsa. Representative cells from SA2 WT-myc or SA2–12xA-myc cell lines after induction with 2 μg/ml doxycycline are shown. Scale bar 10 μm.</p> <p>(B) HeLa cells were induced to express SA2-WT-myc or SA2–12xA-myc by different amounts of doxycycline as indicated, and processed as in (A). More than 50 cells in prometaphase or metaphase were selected randomly from each sample. The distance between sister chromatids was determined for five chromosomes in each cell and averaged. Light gray bars indicate average values that have been measured in one or two cells, and darker gray bars indicate average values that have been measured in three or more cells. Diamonds indicate the average distance for all cells in a given sample.</p> <p>(C) Representative immunofluorescence image of normal anaphase in a cell expressing SA2–12xA-myc. The cell was not extracted prior to fixation, so the soluble pool of SA2–12xA-myc is revealed by myc-staining.</p></div

Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2

<div><p>(A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030069#pbio-0030069-g001" target="_blank">Figure 1</a>C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side.</p> <p>(B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2.</p> <p>(C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).</p></div