Cleavage of DNA with Methidiumpropyl-EDTA

Attachment of ethylenediaminetetraacetate (EDTA) to the DNA intercalator methidium creates an efficient DNA cleaving molecule, methidiumpropyl-EDTA (MPE). MPE·Fe(II) (10-7 M) single strand cleaves supercoiled pBR-322 plasmid DNA (10-5 M) in the presence of O2, converting it to 56% open circular DNA. In the presence of 1 mM dithiothreitol (DTT) and O2, MPE·Fe(II) (10-8 M) converts supercoiled pBR-322 DNA (10-5 M) to 97% open circular and 3% linear DNA. MPE·Mg(II) binds to sonicated calf thymus DNA with a binding affinity of 1.2 x 10-5 M-1 and binding site size of 1.9 base pairs, and unwinds supercoiled PM2 plasmid DNA with an unwinding angle of 11° ± 3°. The reaction conditions for DNA cleavage and factors affecting the cleavage efficiency by MPE·Fe(II) have been determined. The cleavage is dependent on Fe(II) and O2, inhibited by chelating agents, enhanced by reducing agents (ascorbate &gt; DTT &gt; NADH), inhibited by catalase, partially inhibited by radical scavengers, relatively unaffected by sodium concentration, and optimum at pH 7.4 (in Tris·HCl buffer). MPE·Fe(II) cleaves DNA in a relatively non-sequence specific manner, with significantly lower sequence specificity than the enzyme DNAse I, and is a useful footprinting tool for the determination of small molecule binding sites on naturally occurring heterogeneous DNA. The products from the cleavage reaction of MPE·Fe(II) with DNA have been characterized. The results demonstrate that each strand scission produces a free nucleotide base, a 5' phosphoryl group, and a mixture of 3' phosphoryl and 3' phosphoglycolic acid groups left on the polynucleotide chain. Very little malondialdehyde or base-propenals are produced. These products are consistent with the intermediacy of hydroxyl radical in the strand scission reaction.</p

Immunochemotherapy With Obinutuzumab or Rituximab for Previously Untreated Follicular Lymphoma in the GALLIUM Study: Influence of Chemotherapy on Efficacy and Safety

PurposeThe GALLIUM study (ClinicalTrials.gov identifier: NCT01332968) showed that obinutuzumab (GA101;G) significantly prolonged progression-free survival (PFS) in previously untreated patients with follicular lymphoma relative to rituximab (R) when combined with cyclophosphamide (C), doxorubicin, vincristine (V), and prednisone (P;CHOP);CVP;or bendamustine. This report focuses on the impact of chemotherapy backbone on efficacy and safety.Patients and Methods: A total of 1,202 patients with previously untreated follicular lymphoma (grades 1 to 3a), advanced disease (stage III or IV, or stage II with tumor diameter 7 cm), Eastern Cooperative Oncology Group performance status 0 to 2, and requiring treatment were randomly assigned 1:1 to G 1,000 mg on days 1, 8, and 15 of cycle 1 and day 1 of subsequent cycles or R 375 mg/m(2) on day 1 of each cycle, for six to eight cycles, depending on chemotherapy (allocated nonrandomly by center). Responding patients received G or R for 2 years or until disease progression.Results: Baseline Follicular Lymphoma International Prognostic Index risk, bulky disease, and comorbidities differed by chemotherapy. After 41.1 months median follow-up, PFS (primary end point) was superior for G plus chemotherapy (overall hazard ratio [HR], 0.68;95% CI, 0.54 to 0.87;P = .0016), with consistent results across chemotherapy backbones (bendamustine: HR, 0.63;95% CI, 0.46 to 0.88;CHOP: HR, 0.72;95% CI, 0.48 to 1.10;CVP: HR, 0.79;95% CI, 0.42 to 1.47). Grade 3 to 5 adverse events, notably cytopenias, were most frequent with CHOP. Grade 3 to 5 infections and second neoplasms were most frequent with bendamustine, which was associated with marked and prolonged reductions in T-cell counts. Fatal events were more frequent in patients treated with bendamustine, possibly reflecting differences in patient risk profiles.Conclusion: Improved PFS was observed for G plus chemotherapy for all three chemotherapy backbones. Safety profiles differed, although comparisons are confounded by nonrandom chemotherapy allocation

Entanglement Entropy for Singular Surfaces

We study entanglement entropy for regions with a singular boundary in higher dimensions using the AdS/CFT correspondence and find that various singularities make new universal contributions. When the boundary CFT has an even spacetime dimension, we find that the entanglement entropy of a conical surface contains a term quadratic in the logarithm of the UV cut-off. In four dimensions, the coefficient of this contribution is proportional to the central charge 'c'. A conical singularity in an odd number of spacetime dimensions contributes a term proportional to the logarithm of the UV cut-off. We also study the entanglement entropy for various boundary surfaces with extended singularities. In these cases, similar universal terms may appear depending on the dimension and curvature of the singular locus.Comment: 66 pages,4 figures. Some typos are removed and a reference is adde

Casimir forces between cylinders and plates

We study collective interaction effects that result from the change of free quantum electrodynamic field fluctuations by one- and two-dimensional perfect metal structures. The Casimir interactions in geometries containing plates and cylinders is explicitly computed using partial wave expansions of constrained path integrals. We generalize previously obtained results and provide a more detailed description of the technical aspects of the approach \cite{Emig06}. We find that the interactions involving cylinders have a weak logarithmic dependence on the cylinder radius, reflecting that one-dimensional perturbations are marginally relevant in 4D space-time. For geometries containing two cylinders and one or two plates, we confirm a previously found non-monotonic dependence of the interaction on the object's separations which does not follow from pair-wise summation of two-body forces. Qualitatively, this effect is explained in terms of fluctuating charges and currents and their mirror images

Some Calculable Contributions to Holographic Entanglement Entropy

Using the AdS/CFT correspondence, we examine entanglement entropy for a boundary theory deformed by a relevant operator and establish two results. The first is that if there is a contribution which is logarithmic in the UV cut-off, then the coefficient of this term is independent of the state of the boundary theory. In fact, the same is true of all of the coefficients of contributions which diverge as some power of the UV cut-off. Secondly, we show that the relevant deformation introduces new logarithmic contributions to the entanglement entropy. The form of some of these new contributions is similar to that found recently in an investigation of entanglement entropy in a free massive scalar field theory [1].Comment: 52 pages, no figure

Cleavage of double helical DNA by methidium-propyl-EDTA-iron(II)

We report the synthesis of a simple bifunctional molecule, methidiumpropyl-EDTA (MPE) (1), which contains the DNA intercalator methidium covalently bound by a short hydrocarbon tether to the metal chelator EDT A. In the presence of ferrous ion and oxygen this reagent efficiently produces single-strand breaks and some double-strand breaks in double helical DNA

Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE•Ni(II), and MPE•Mg(II) to calf thymus DNA are 2.4 X 10^4 M^(-1), 1.5 X 10^5 M^(-1), and 1.2 X 10^5 M^-1), respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE•Mg(II) unwinds PM2 DNA 11 ± 3° per bound molecule. MPE•Fe(II) in the presence of O_2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate > dithiothreitol > NADPH. At concentrations of 0.1-0.01 µM in MPE•Fe(II) and 10 µM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 µM in base pairs) with MPE•Fe(II) (0.1-0.01 µM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HC1 buffer). The optimum cleavage time is 3.5 h (22 °C). DNA cleavage is efficient in a Na^+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE•Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE•Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphogiycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical