Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells

Cellular recovery from ionizing radiation (IR)-induced damage involves poly(ADP-ribose) polymerase (PARP-1 and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle microbeam delivering 3.2 MeV protons from a Van de Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs to local irradiation, concomitant with the recruitment of ATM and Rad51 at sites of DNA damage, both proteins being involved in DNA strand break repair. We found a co-localization but no connection between two DNA damage-dependent post-translational modifications, namely poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however, should be considered and used as bona fide immediate sensitive markers of IR damage in living cells. This technique thus provides a powerful approach aimed at understanding the interactions between the signals originating from sites of DNA damage and the subsequent activation of DNA strand break repair mechanisms.

An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis

The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.

Functional analysis of the glycero-manno-heptose 7-phosphate kinase domain from the bifunctional HldE protein, which is involved in ADP-L-glycero-D-manno-heptose biosynthesis

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In Escherichia coli, the inner core consists of two 3-deoxy-d-manno-octulosonic acid and three glycero-manno-heptose residues. The HldE protein participates in the biosynthesis of ADP-glycero-manno-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the E. coli ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.

Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.

The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.

Synthesis of alkylcarbonate analogs of O-acetyl-ADP-ribose

The non-hydrolyzable alkylcarbonate analogs of O-acetyl-ADP-ribose have been synthesized from the phosphorylated ribose derivatives after coupling with AMP morpholidate promoted by mechanical grinding. The analogs were assessed for their ability to inhibit the human sirtuin homolog SIRT1. © 2013 The Royal Society of Chemistry.

A review of ADP processing software

Data processing is an essential part of Acoustic Doppler Profiler (ADP) surveys, which have become the standard tool in assessing flow characteristics at tidal power development sites. In most cases, further processing beyond the capabilities of the manufacturer provided software tools is required. These additional tasks are often implemented by every user in mathematical toolboxes like MATLAB, Octave or Python. This requires the transfer of the data from one system to another and thus increases the possibility of errors. The application of dedicated tools for visualisation of flow or geographic data is also often beneficial and a wide range of tools are freely available, though again problems arise from the necessity of transferring the data. Furthermore, almost exclusively PCs are supported directly by the ADP manufacturers, whereas small computing solutions like tablet computers, often running Android or Linux operating systems, seem better suited for online monitoring or data acquisition in field conditions. While many manufacturers offer support for developers, any solution is limited to a single device of a single manufacturer. A common data format for all ADP data would allow development of applications and quicker distribution of new post processing methodologies across the industry.

Current and Turbulence Measurement with Collocated ADP and Turbulence Profiler Data

This paper presents a current and turbulence measurement campaign conducted at a test site in an energetic tidal channel known as Strangford Narrows, Northern Ireland. The data was collected as part of the MaRINET project funded by the EU under their FP7 framework. It was a collaborative effort between Queen’s University Belfast, SCHOTTEL and Fraunhofer IWES. The site is highly turbulent with a strong shear flow. Longer term measurements of the flow regime were made using a bottom mounted Acoustic Doppler Profiler (ADP). During a specific turbulence measurement campaign, two collocated in- struments were used to measure incoming flow characteristics: an ADP (Aquadopp, Nortek) and a turbulence profiler (MicroRider, Rockland Scientific International). The instruments recorded the same incoming flow, so that direct comparisons between the data can be made. In this study the methodology adopted to deploy the instruments is presented. The resulting turbulence measurements using the different types of instrumentation are compared and the usefulness of each instrument for the relevant range of applications is discussed. The paper shows the ranges of the frequency spectra obtained using the different instruments, with the combined measurements providing insight into the structure of the turbulence across a wide range of scales.

Doxorubicin induces the cleavage of Poly (ADP-ribose) polymerase, a marker of programmed cell death (apoptosis) in mouse cardiomyocytes

Cancer is one of the leading causes of death in the world. Despite this, a growing number of people are surviving the disease due to medical advancements and the development of numerous new therapies. Doxorubicin, a chemotherapeutic agent, is a widely-used and successful first-line anti-tumour treatment. However, the established toxic and deleterious effects of the drug on the cardiovascular system confer increased risk of congestive heart failure, thereby necessitating the use of reduced doxorubicin doses. In order to investigate how these events are initiated, mouse cardiomyocytes (HL-1) were treated in vitro with varying concentrations of doxorubicin (0.5-4.0 µmol/L). Following treatment (24h), a marked level of cell death was observed in comparison to untreated cardiomyocytes; the level of death appeared to correlate with the concentration of the drug used. Western blotting revealed the cleavage of full length Poly (ADP-ribose) polymerase (PARP) into 89 and 24kDa fragments, a process which is instrumental in triggering programmed cell death/apoptosis. Importantly, results suggested that this event may be independent of caspase 3 cleavage and thus activation. A number of previous studies have reported a functional role for both Mitofusin-2 (Mfn2) and NADPH oxidase 2 (Nox2) in the cardiotoxic response. Given that PARP cleavage is a validated indicator of cellular apoptosis, these results clearly indicate that this marker could be used in future studies when determining if depletion of the above proteins would cause a reduction in or eradicate the pro-apoptotic action of this agent on cardiomyocytes. Such investigations may lead to significant developments in ensuring that doxorubicin can achieve its full therapeutic anti-tumour potential without causing the subsequent deleterious effects on the cardiovascular system.

Clinical Application of Poly(ADP-Ribose) Polymerase Inhibitors in High-Grade Serous Ovarian Cancer

: High-grade serous ovarian cancer is characterized by genomic instability, with one half of all tumors displaying defects in the important DNA repair pathway of homologous recombination. Given the action of poly(ADP-ribose) polymerase (PARP) inhibitors in targeting tumors with deficiencies in this repair pathway by loss of BRCA1/2, ovarian tumors could be an attractive population for clinical application of this therapy. PARP inhibitors have moved into clinical practice in the past few years, with approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA) within the past 2 years. The U.S. FDA approval of olaparib applies to fourth line treatment in germline BRCA-mutant ovarian cancer, and European EMA approval to olaparib maintenance in both germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. In order to widen the ovarian cancer patient population that would benefit from PARP inhibitors, predictive biomarkers based on a clear understanding of the mechanism of action are required. Additionally, a better understanding of the toxicity profile is needed if PARP inhibitors are to be used in the curative, rather than the palliative, setting. We reviewed the development of PARP inhibitors in phase I-III clinical trials, including combination trials of PARP inhibitors and chemotherapy/antiangiogenics, the approval for these agents, the mechanisms of resistance, and the outstanding issues, including the development of biomarkers and the rate of long-term hematologic toxicities with these agents.

IMPLICATIONS FOR PRACTICE: The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib has recently received approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA), with a second agent (rucaparib) likely to be approved in the near future. However, the patient population with potential benefit from PARP inhibitors is likely wider than that of germline BRCA mutation-associated disease, and biomarkers are in development to enable the selection of patients with the potential for clinical benefit from these agents. Questions remain regarding the toxicities of PARP inhibitors, limiting the use of these agents in the prophylactic or adjuvant setting until more information is available. The indications for olaparib as indicated by the FDA and EMA are reviewed.

Synthesis and Binding of Stable Bisubstrate Ligands for Phosphoglycerate Kinase

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesised with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity - in accordance with its approximation to the transition state for the reaction catalysed by PGK.

Isopolar phosphonate analogue of adenosine diphosphate ribose

The synthesis of the bisphosphonate ADP-ribose, in which acetylene has replaced the oxygen of the pyrophosphate linkage, is reported.

BRCA1 Functions as a Differential Modulator of Chemotherapy-induced Apoptosis

We have evaluated the role played by BRCA1 in mediating the phenotypic response to a range of chemotherapeutic agents commonly used in cancer treatment. Here we provide evidence that BRCA1 functions as a differential mediator of chemotherapy-induced apoptosis. Specifically, we demonstrate that BRCA1 mediates sensitivity to apoptosis induced by antimicrotubule agents but conversely induces resistance to DNA-damaging agents. These data are supported by a variety of experimental models including cells with inducible expression of BRCA1, siRNA-mediated inactivation of endogenous BRCA1, and reconstitution of BRCA1-deficient cells with wild-type BRCA1. Most notably we demonstrate that BRCA1 induces a 10–1000-fold increase in resistance to a range of DNA-damaging agents, in particular those that give rise to double-strand breaks such as etoposide or bleomycin. In contrast, BRCA1 induces a >1000-fold increase in sensitivity to the spindle poisons, paclitaxel and vinorelbine. Fluorescence-activated cell sorter analysis demonstrated that BRCA1 mediates G2/M arrest in response to both antimicrotubule and DNA-damaging agents. However, poly(ADP-ribose) polymerase and caspase-3 cleavage assays indicate that the differential effect mediated by BRCA1 in response to these agents occurs through the inhibition or induction of apoptosis. Therefore, our data suggest that BRCA1 acts as a differential modulator of apoptosis depending on the nature of the cellular insult.

Epidermal Growth Factor Receptor Activity Determines Response of Colorectal Cancer Cells to Gefitinib Alone and in Combination with Chemotherapy.

Purpose: Up to now, there have been no established predictive markers for response to epidermal growth factor receptor (EGFR/HER1/erbB1) inhibitors alone and in combination with chemotherapy in colorectal cancer. To identify markers that predict response to EGFR-based chemotherapy regimens, we analyzed the response of human colorectal cancer cell lines to the EGFR-tyrosine kinase inhibitor, gefitinib (Iressa, AstraZeneca, Wilmington, DE), as a single agent and in combination with oxaliplatin and 5-fluorouracil (5-FU). Experimental Design: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays and analyzed by ANOVA. Apoptosis was measured by flow cytometry, poly(ADP-ribose) polymerase, and caspase 3 cleavage. EGFR protein phosphorylation was detected by Western blotting. Results: Cell lines displaying high constitutive EGFR phosphorylation (a surrogate marker for EGFR activity) were more sensitive to gefitinib. Furthermore, in cell lines exhibiting low constitutive EGFR phosphorylation, an antagonistic interaction between gefitinib and oxaliplatin was observed, whereas in cell lines with high basal EGFR phosphorylation, the interaction was synergistic. In addition, oxaliplatin treatment increased EGFR phosphorylation in those cell lines in which oxaliplatin and gefitinib were synergistic but down-regulated EGFR phosphorylation in those lines in which oxaliplatin and gefitinib were antagonistic. In contrast to oxaliplatin, 5-FU treatment increased EGFR phosphorylation in all cell lines and this correlated with synergistic decreases in cell viability when 5-FU was combined with gefitinib. Conclusions: These results suggest that phospho-EGFR levels determine the sensitivity of colorectal cancer cells to gefitinib alone and that chemotherapy-mediated changes in phospho-EGFR levels determine the nature of interaction between gefitinib and chemotherapy.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.