SUMOylation of TopoIIα inhibits its decatenation activity, preventing resolution of tangled DNA at centromeres before anaphase onset.

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.

Arabidopsis (Arabidopsis thaliana) genome contains more than 90 armadillo (arm) repeat proteins. However, their functions are largely unknown. Here, we report that an Arabidopsis arm repeat protein is involved in abscisic acid (ABA) response. We carried out two-hybrid screens to identify signaling components that modulate ABA-responsive gene expression. Employing a transcription factor, ABF2, which controls the ABA-dependent gene expression via the G-box type ABA-responsive elements, we isolated an arm repeat protein. The ABF2-interacting protein, designated as ARIA (arm repeat protein interacting with ABF2), has another conserved sequence motif, BTB/POZ (broad complex, tramtrak, and bric-a-brac/poxvirus and zinc finger) domain, in the C-terminal region. The physiological relevance of ABF2-ARIA interaction was supported by their similar expression patterns and similar subcellular localization. Plants overexpressing ARIA are hypersensitive to ABA and high osmolarity during germination and insensitive to salt during subsequent seedling growth. By contrast, an ARIA knockout mutant exhibits ABA and glucose insensitivities. Changes in the expression levels of several ABF2-regulated genes were also observed in ARIA overexpression lines, indicating that ARIA modulates the transcriptional activity of ABF2. Together, our data indicate that ARIA is a positive regulator of ABA response.Armadillo (arm) repeat is a 42-amino acid proteinprotein interaction motif (Peifer et al., 1994;Hatzfeld, 1999;Andrade et al., 2001). The repeat was first identified in the Drosophila segment polarity gene armadillo (Riggleman et al., 1989) and since then in many eukaryotic proteins involved in cell signaling or cellular architecture. Armadillo and its vertebrate homolog b-catenin are components of the Wingless and the Wnt signaling pathways, which determine the patterning of Drosophila embryo body segments and vertebrate cell fates, respectively (Polakis, 2000). When triggered by the Wingless or Wnt growth factor signal, otherwise unstable Armadillo/b-catenin becomes stabilized, translocates into the nucleus, and together with the TCF/LEF subfamily of transcription factors, activates the Wingless/Wnt target genes. b-Catenin also plays a structural role in cell-cell adhesion by linking the transmembrane adhesion molecules cadherins to actin cytoskeleton.Pfam (http://www.sanger.ac.uk/Software/Pfam/) and SMART (http://smart.embl-heidelberg.de/) protein databases enlist more than 90 Arabidopsis (Arabidopsis thaliana) arm repeat proteins. Based on their sequence homology, these proteins can be grouped into several different subfamilies such as impotin-a, kinesin, and U-box protein families (Coates, 2003). However, the functions of the Arabidopsis and other plant arm repeat proteins have not been characterized in detail except those of ARC1 and PHOR1. ARC1 interacts with an S-locus receptor kinase of Brassica (Gu et al., 1998) and has been demonstrated to be a positive regulator of the self-incompatibility response (Stone et al., 1999). A recent ...

BackgroundThis study aims to assess both feasibility and accuracy of renal dosimetry imaging protocols in patients receiving Lutate therapy for neuroendocrine tumours (NETs), when data acquisition over multiple days is not possible on all cycles.MethodPatients who had received a full 4 cycles of Lutate therapy with complete imaging at each cycle were included. Imaging consisted of quantitative SPECT/CT of the kidneys at 4, 24 and 96–120 h post injection. Renal absorbed dose was calculated for each data set, and in addition, five alternative methods were explored for comparison. Method 1: a patient average clearance time (t1/2 average) derived from the first half of contributing patient data was used to estimate absorbed dose for subsequent patients based on 4 h imaging alone; method 2: t1/2 average was applied to subsequent patients on 24 h imaging alone; method 3: a patient-specific clearance rate (t1/2 patient) was determined from complete image data of cycle 1 and applied subsequently to remaining cycles using 4 h image data alone; method 4: t1/2 patient was applied to 24 h imaging alone in subsequent cycles; method 5: the 120 h data was estimated on subsequent cycles based on the cycle 1 fraction of injected activity (%IA) at 24 and 120 h.ResultsTwenty treatments from 18 patients, resulting in 80 cycles of therapy, were analysed. The measured average renal absorbed dose per cycle of treatment was 0.38 ± 0.19 Gy/GBq when derived from full imaging data. The use of t1/2 average applied to a single time point led to large deviations of dose estimates from true values (on average 59% and 30%, when using 4 h data and 24 h data, respectively). The use of complete image data on cycle 1 and the derivation of t1/2 patient led to improved dose estimates, with an average deviation from true values of 13% and 2% when using 4 h data only and 24 h data only, respectively. The use of a 120 h %IA derived from cycle 1 led to an average deviation from true dose estimates of 14%.ConclusionIn instances where demands on both patients and facilities make multiple time point data acquisition impractical, renal dosimetry is best derived through complete imaging at cycle 1 only followed by a single 24 h imaging time point on subsequent cycles, assuming no significant changes in renal function during the time course of therapy.

BackgroundDose rate variation is a critical factor affecting radionuclide therapy (RNT) efficacy. Relatively few studies to date have investigated the dose rate effect in RNT. Therefore, the aim of this study was to benchmark 90Y RNT (at different dose rates) against external beam radiotherapy (EBRT) in vitro and compare cell kill responses between the two irradiation processes.ResultsThree human colorectal carcinoma (CRC) cell lines (HT29, HCT116, SW48) were exposed to 90Y doses in the ranges 1–10.4 and 6.2–62.3 Gy with initial dose rates of 0.013–0.13 Gy/hr (low dose rate, LDR) and 0.077–0.77 Gy/hr (high dose rate, HDR), respectively. Results were compared to a 6-MV photon beam doses in the range from 1–9 Gy with constant dose rate of 277 Gy/hr. The cell survival parameters from the linear quadratic (LQ) model were determined. Additionally, Monte Carlo simulations were performed to calculate the average dose, dose rate and the number of hits in the cell nucleus.For the HT29 cell line, which was the most radioresistant, the α/β ratio was found to be ≈ 31 for HDR–90Y and ≈ 3.5 for EBRT. LDR–90Y resulting in insignificant cell death compared to HDR–90Y and EBRT. Simulation results also showed for LDR–90Y, for doses ≲ 3 Gy, the average number of hits per cell nucleus is ≲ 2 indicating insufficiently delivered lethal dose. For 90Y doses  3 Gy the number of hits per nucleus decreases rapidly and falls below ≈ 2 after ≈ 5 days of incubation time. Therefore, our results demonstrate that LDR–90Y is radiobiologically less effective than EBRT. However, HDR–90Y at ≈ 56 Gy was found to be radiobiologically as effective as acute ≈ 8 Gy EBRT.ConclusionThese results demonstrate that the efficacy of RNT is dependent on the initial dose rate at which radiation is delivered. Therefore, for a relatively long half-life radionuclide such as 90Y, a higher initial activity is required to achieve an outcome as effective as EBRT.

Organo-layered double hydroxide/polypropylene (LDH/PP) nanocomposites were successfully synthesized via a solution blending method. As an attempt to improve the compatibility with hydrophobic PP, the LDH surface was modified by the incorporation of various anionic surfactants via electrostatic interaction with LDH cationic layers. Surfactants were selected by considering the aliphatic carbon chain length (laurate, palmitate, stearate and dodecyl sulfate) and anionic functional groups (-COO À , -OPO 3 2À , and -OSO 3 À ) with the purpose of optimizing the homogeneous dispersion in the PP matrix. In PP nanocomposites containing LDH modified with alkyl carboxylate, the (00l) X-ray diffraction (XRD) peaks originating from organo-LDH were not observed, indicating that organo-LDH layers were fully exfoliated and homogeneously dispersed within the PP matrix, which were also confirmed by cross-sectional TEM analysis. However, PP nanocomposites containing LDH modified with dodecyl sulfate and lauryl phosphate showed broad (00l) XRD peaks, indicating that organo-LDH was partially exfoliated.According to the thermogravimetric analysis, the thermal stability (T 0.5 ) of organo-LDH/PP nanocomposites was significantly improved by 37-60 K, depending on the type and loading content of organo-LDH compared to that of pristine PP. PP nanocomposites containing well-dispersed organo-LDH showed substantial enhancement of the elastic modulus with little decrease of tensile strength.These results are due to the increased interface volume fraction provided by the exfoliated LDH nanosheets.

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.

Background: Mitotic SUMOylation is critical for faithful chromosome segregation in eukaryotes. Results: PICH (Plk1-interacting checkpoint helicase) is a SUMO-interacting protein and a mitotic SUMO substrate. Conclusion: PICH can be regulated by binding to SUMOylated proteins and through its own SUMOylation at mitotic centromeres. Significance: The regulation of PICH by SUMO interaction and SUMOylation reveals a novel role for mitotic SUMOylation in centromeric chromatin organization.