A comparison of the binding of metal complexes to duplex and quadruplex DNA

Electrospray ionisation mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to compare the binding of mononuclear nickel, ruthenium and platinum complexes to double stranded DNA (dsDNA) and quadruplex DNA (qDNA). CD studies provided evidence for the binding of intact complexes of all three metal ions to qDNA. ESI mass spectra of solutions containing platinum or ruthenium complexes and qDNA showed evidence for the formation of non-covalent complexes consisting of intact metal molecules bound to DNA. However, the corresponding spectra of solutions containing nickel complexes principally contained ions consisting of fragments of the initial nickel molecule bound to qDNA. In contrast ESI mass spectra of solutions containing nickel, ruthenium or platinum complexes and dsDNA only showed the presence of ions attributable to intact metal molecules bound to DNA. The fragmentation observed in mass spectral studies of solutions containing nickel complexes and qDNA is attributable to the lower thermodynamic stability of the former metal complexes relative to those containing platinum or ruthenium, as well as the slightly harsher instrumental conditions required to obtain spectra of qDNA. This conclusion is supported by the results of tandem mass spectral studies, which showed that ions consisting of intact nickel complexes bound to qDNA readily undergo fragmentation by loss of one of the ligands initially bound to the metal. The ESI-MS results also demonstrate that the binding affinity of each of the platinum and ruthenium complexes towards qDNA is significantly less than that towards dsDNA.     

Topical treatment with OGG1 enzyme affects UVB-induced skin carcinogenesis

Nonmelanoma skin cancer resulting from UVB exposure is a large and growing problem in the United States. Production of reactive oxygen species (ROS) during the UVB-induced inflammatory response results in the formation of oxidative DNA adducts such as 8-hydroxy-2-deoxyguanine (8-oxo-dG), which have been shown to contribute to the development of this cancer. The 8-oxoguanine DNA glycosylase (OGG1) enzyme repairs 8-oxo-dG adducts, suggesting that enhancing its activity in the skin might increase 8-oxo-dG repair thus preventing skin cancer development. We therefore used the SKH-1 murine model to examine the effect of topically applied OGG1 on UVB-induced skin cancer development. Mice were exposed three times weekly to UVB followed immediately by topical treatment with a formulation of liposome-encapsulated OGG1 enzyme for 25 weeks. While this treatment did not affect UVB-induced tumor multiplicity, it did reduce tumor size and dramatically reduced tumor progression, as indicated by tumor grade. These results suggest that oxidative DNA damage contributes to the progression of UVB-induced skin tumors and that a topical formulation containing OGG1, perhaps in conjunction with other DNA repair enzymes such as T4 endonuclease V, could be used in populations at high risk for skin cancer development.     

Tracking regression and progression of atherosclerosis in human carotid arteries using high-resolution magnetic resonance imaging

BACKGROUND AND PURPOSE: Magnetic resonance imaging (MRI) can accurately and reproducibly measure the volume of atherosclerotic plaque in human carotid arteries. Atherosclerotic plaques may either progress or regress over time, depending on individual risk factors and treatment regimens. This study was designed to determine if regression or progression of human carotid atherosclerosis in patients receiving statin therapy over 24 months can be detected by high-resolution MRI. METHODS: In 11 subjects who had undergone unilateral carotid endarterectomy and were on statin therapy, volumes for total carotid artery, concentric wall (normal wall), eccentric wall (plaque), and lumen were quantified at 0, 16 and 24 months using a 1.5-T human imager equipped with 6-cm phased array coils. RESULTS: The interobserver mean coefficient of variation (CV) was lowest for the lumen volume (3.1%) and highest for the plaque volume (9.8%). The interscan mean CV was lowest for the total artery volume (3.2%) and highest for the plaque volume (9.9%). As much as 26% regression and 35% progression were observed in individual subject's carotid artery eccentric wall (plaque) volumes over time. Mean eccentric wall volume increased 5% by 16 months and 8% by 24 months. Mean total wall volume increased slightly at both 16 and 24 months (+1.2% and +1.8%). CONCLUSIONS: High-resolution MRI provides a noninvasive reproducible method of tracking changes in carotid atherosclerosis. This pilot study detected changes in individual subjects at both 16 and 24 months. MRI tracking of changes in atherosclerotic plaques should prove useful in assessing vascular disease risk and monitoring the efficacy of interventions designed to induce regression or retard progression.     

Growth mechanism of textured MgO thin films via SSCVD

Magnesium oxide thin films have been deposited with use of single source chemical vapor deposition (SSCVD). The resultant films were examined by using transmission electron microscopy, X-ray texture analysis, and pole figure analysis. Due to the nature of the chemical reactions occurring at the surface during SSCVD growth, which result in a high growth rate/low flux environment, films of (111) orientation have been achieved without an amorphous underlayer, an unusual result for films of this orientation. Moreover the films have a strong degree of biaxial texturing in the x-y plane as found with X-ray texture analysis. These findings have important implications for buffer layers in perovskite thin film devices. The mechanism producing these structures has been revealed by using TEM and is discussed here.     

Synthesis, DNA polymerase incorporation, and enzymatic phosphate hydrolysis of formamidopyrimidine nucleoside triphosphates

The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.     

Silicon carbide passive heating elements in microwave-assisted organic synthesis

Microwave-assisted organic synthesis in nonpolar solvents is investigated utilizing cylinders of sintered silicon carbide (SiC)--a chemically inert and strongly microwave absorbing material--as passive heating elements (PHEs). These heating inserts absorb microwave energy and subsequently transfer the generated thermal energy via conduction phenomena to the reaction mixture. The use of passive heating elements allows otherwise microwave transparent or poorly absorbing solvents such as hexane, carbon tetrachloride, tetrahydrofuran, dioxane, or toluene to be effectively heated to temperatures far above their boiling points (200-250 degrees C) under sealed vessel microwave conditions. This opens up the possibility to perform microwave synthesis in unpolar solvent environments as demonstrated successfully for several organic transformations, such as Claisen rearrangements, Diels-Alder reactions, Michael additions, N-alkylations, and Dimroth rearrangements. This noninvasive technique is a particularly valuable tool in cases where other options to increase the microwave absorbance of the reaction medium, such as the addition of ionic liquids as heating aids, are not feasible due to an incompatibility of the ionic liquid with a particular substrate. The SiC heating elements are thermally and chemically resistant to 1500 degrees C and compatible with any solvent or reagent.     

Evaluation of electron-withdrawing group effects on heme binding in designed proteins: implications for heme a in cytochrome c oxidase

Heme a, the metalloporphyrin cofactor unique to cytochrome c oxidases, differs from the more common heme b by two chemical modifications, a C-2 hydroxyethylfarnesyl group and a C-8 formyl group. To elucidate a role of the C-8 formyl group, we compare the heme affinity, spectroscopy, and electrochemistry of a heme a mimic, Fe(diacetyldeuterioporphyrin IX) or Fe(DADPIX), with heme b, Fe(protoporphryrin IX) or Fe(PPIX), incorporated into a designed heme protein. The [Delta7-H3m]2 protein ligand, or maquette, selected for this study contains two equivalent bis-(3-methyl-L-histidine) heme binding sites within a four-alpha-helix bundle scaffold. The spectroscopic data on Fe(PPIX) and Fe(DADPIX) bound to [Delta7-H3m]2 demonstrate that these complexes are excellent synthetic analogues for natural cytochromes b and a, respectively. Comparison of the spectroscopic, electrochemical, and equilibrium thermodynamic data measured for the Fe(PPIX)-[Delta7-H3m]2 maquette with the previously reported Fe(PPIX)-[Delta7-His]2 complex demonstrates that changing the heme axial ligands to 3-methyl-L-histidine from L-histidine does not alter the resulting heme protein properties significantly in either oxidation state. Heme binding studies demonstrate that [Delta7-H3m]2 binds two ferrous Fe(DADPIX) or Fe(PPIX) moieties with similar dissociation constant values. However, in the ferric state, the data show that [Delta7-H3m]2 only binds a single Fe(DADPIX) and that one 2500-fold weaker than oxidized Fe(PPIX). The data demonstrate that the 4.6 kcal mol(-1) weakened affinity of [Delta7-H3m]2 for oxidized Fe(DADPIX) results in the majority of the 160 mV, 3.7 kcal mol(-1), positive shift in the heme reduction potential relative to Fe(PPIX). These data indicate that a role of the formyl group on heme a is to raise the iron reduction potential, thus making it a better electron acceptor, but that it does so by destabilizing the affinity of bis-imidazole sites for the ferric state.     

A lattice model of vitrification and gelation

In this work we introduce a simple lattice model with T-shaped molecules in two dimensions that exhibits a rich range of morphological behaviors. Depending on the volume fraction and quench path, this system can adopt uniform liquid, solution, and phase-separated states, as well as inhomogeneous glass or gel-like states, as revealed by dynamic mean-field simulations. An important characteristic of this system is the existence of a large number of degenerate low-energy states with small barriers that leads to a broad, kinetically explored landscape. The mean-field stability and phase diagram of this model is constructed and provides a useful guide for understanding the complex behaviors of the system. One striking feature is that there is a cascade of instabilities that converge to mark the onset of what we identify as the glass transition. Both dynamic mean-field and Monte Carlo simulations reveal glass-like relaxation dynamics. Our results lead to a picture of gelation as a continuation of the glass transition into the two-phase region, or equivalently, as an incomplete phase separation arrested by the onset of the glass transition.     

Tight binding inhibitors of N-acyl amino sugar and N-acyl amino acid deacetylases

Very potent inhibitors were synthesized for the enzymatic deacetylation of N-acetyl-d-glucosamine-6-phosphate (NagA) and N-acetyl-d-glutamate (DGD). The methyl phosphonamidate derivative of d-glucosamine-6-phosphate bound to N-acetyl-d-glucosamine-6-phosphate deacetylase with an equilibrium dissociation constant of 34 +/- 5 nM at pH 7.5 and an association rate constant of 6.1 x 103 M-1 s-1. The inhibition constant is 4000-fold lower than the Michaelis constant for the substrate N-acetyl-d-glucosamine-6-phosphate. N-Acetyl-d-glutamate deacetylase was inhibited by the methyl phosphonamidate derivative of d-glutamate with an inhibition constant of 460 +/- 70 pM at pH 7.6. The inhibitor bound to the enzyme 500 000-fold tighter than the Michaelis constant for N-formyl-d-glutamate. These compounds mimic the putative tetrahedral intermediate formed upon nucleophilic attack of an activated water molecule on the amide bond of the target substrate. These inhibitors should prove useful in the elucidation of the enzyme-substrate interactions for enzymes within the amidohydrolase superfamily.     

Synthesis and Investigation of Advanced Energetic Materials Based on Bispyrazolylmethanes

Herein we present the preparation and characterization of three new bispyrazolyl‐based energetic compounds with great potential as explosive materials. The reaction of sodium 4‐amino‐3,5‐dinitropyrazolate ( 5 ) with dimethyl iodide yielded bis(4‐amino‐3,5‐dinitropyrazolyl)methane ( 6 ), which is a secondary explosive with high heat resistance (T_dec=310 °C). The oxidation of this compound afforded bis(3,4,5‐trinitropyrazolyl)methane ( 7 ), which is a combined nitrogen‐ and oxygen‐rich secondary explosive with very high theoretical and estimated experimental detonation performance (V_det (theor)=9304 m s^?1 versus V_det(exp)=9910 m s^?1) in the range of that of CL‐20. Also, the thermal stability (T_dec=205 °C) and sensitivities of 7 are auspicious. The reaction of 6 with in situ generated nitrous acid yielded the primary explosive bis(4‐diazo‐5‐nitro‐3‐oxopyrazolyl)methane ( 8 ), which showed superior properties to those of currently used diazodinitrophenol (DDNP).