ENHANCEMENT OF ULTRAVIOLET-DNA REPAIR IN den V GENE TRANSFECTANTS and T4 ENDONUCLEASE V-LIPOSOME RECIPIENTS

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.     

Visual immunoprecipitate assay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.09): collaborative study

AOAC Official Method 996.09, Visual Immunoprecipitate Assay (VIP) for Escherichia coli O157:H7, was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by VIP assay and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment procedure and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 15 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level, where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 396 test portions and controls by both methods, for a total of 792 test portions. Of the 396 paired test portions, 75 were positive and 230 were negative by both the VIP and culture methods. Eleven test portions were presumptively positive by VIP and could not be confirmed culturally; 32 were negative by VIP, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the VIP method. There was no statistical difference between results obtained with the VIP for EHEC 8 h method and the culture method except for cooked beef, where the VIP had significantly higher recovery for one inoculation level.     

UV-DNA Damage in Mouse and Human Cells Induces the Expression of Tumor Necrosis Factor α

Ultraviolet light induces the expression of tumor necrosis factor α (TNFα) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V in-creased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNFα expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNFa gene expression in mouse and human cells.     

Competition and selectivity in the reaction of nitriles on ge(100)-2x1

We have experimentally investigated bonding of the nitrile functional group (R-Ctbd1;N:) on the Ge(100)-2x1 surface with multiple internal reflection infrared spectroscopy. Density functional theory calculations are used to help explain trends in the data. Several probe molecules, including acetonitrile, 2-propenenitrile, 3-butenenitrile, and 4-pentenenitrile, were studied to elucidate the factors controlling selectivity and competition on this surface. It is found that acetonitrile does not react on the Ge(100)-2x1 surface at room temperature, a result that can be understood with thermodynamic and kinetic arguments. A [4+2] cycloaddition product through the conjugated pi system and a [2+2] C=C cycloaddition product through the alkene are found to be the dominant surface adducts for the multifunctional molecule 2-propenenitrile. These two surface products are evidenced, respectively, by an extremely intense nu(C=C=N), or ketenimine stretch, at 1954 cm(-)(1) and the nu(Ctbd1;N) stretch near 2210 cm(-)(1). While the non-conjugated molecules 3-butenenitrile and 4-pentenenitrile are not expected to form a [4+2] cycloaddition product, both show vibrational modes near 1954 cm(-)(1). Additional investigation suggests that 3-butenenitrile can isomerize to 2-butenenitrile, a conjugated nitrile, before introduction into the vacuum chamber, explaining the presence of the vibrational modes near 1954 cm(-)(1). Pathways directly involving only the nitrile functional group are thermodynamically unfavorable at room temperature on Ge(100)-2x1, demonstrating that this functional group may prove useful as a vacuum-compatible protecting group.     

Methacrylate and epoxy functionalized nanocomposites based on silsesquioxane cores for use in dental applications

The aim of this study was to design and develop nanocomposites based on silsesquioxane cores (SSQ) with reduced shrinkage properties upon curing for application in dental restorative materials. SSQ based nanocomposites with varying amounts of methacrylate and/or epoxide groups were prepared via Pt-catalyzed hydrosilylation of di(propylene glycol) allyl ether methacrylate, propargyl methacrylate, and 4-vinyl-cyclohexene epoxide combinations with (HMe₂SiOSiO_1.5)₈. The compounds were obtained in high yield (>85%) and were readily characterized by NMR, FTIR, GPC, DSC and TGA. The resultant nanocomposites were clear, viscous liquids at room temperature with working viscosities well suited for dental composites. Initial results show these materials have significantly lower shrinkage during curing than traditional monomers used for dental composite applications.     

Equivalence of Visual Immunoprecipitate Assay (VIP) for Salmonella for the detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study

Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Visual Immunoprecipitate Assay (VIP) for Salmonella and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the VIP method and one by the AOAC culture method. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between VIP for Salmonella interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The method was adopted Official First Action status by AOAC INTERNATIONAL.     

Regioselective synthesis of benzimidazole thiophene inhibitors of polo-like kinase 1

A regioselective synthesis of novel 1-(2-thienyl)-benzimidazole inhibitors of polo-like kinase 1 is described. Amination of substituted 2-iodo or -bromo nitrobenzenes with a 2-aminothiophene derivative catalyzed by Pd₂dba₃ and XANTPHOS in the presence of excess Cs₂CO₃ afforded good yields of the coupled products. Subsequent reduction and cyclization of these intermediates provided the desired benzimidazole compounds.     

The role of exchange-correlation functionals in the potential energy surface and dynamics of N(2) dissociation on W surfaces

We study the dissociative adsorption of N(2) on W(100) and W(110) by means of density functional theory and classical dynamics. Working with a full six-dimensional adiabatic potential energy surface (PES), we find that the theoretical results of the dynamical problem strongly depend on the choice of approximate exchange-correlation functional for the determination of the PES. We consider the Perdew-Wang-91 [Perdew et al., Phys. Rev. B 46, 6671 (1992)] and Perdew-Burke-Ernzerhof (RPBE) [Hammer et al., Phys. Rev. B 59, 7413 (1999)] functionals and carry out a systematic comparison between the dynamics determined by the respective PESs. Even though it has been shown in earlier works that the RPBE may provide better values for the chemisorption energies, our study brings evidence that it gives rise to a PES with excessive repulsion far from the surface.     

Pyrazolo[1,5-a]pyridines as p38 kinase inhibitors

[reaction: see text] A convergent synthesis of substituted pyrazolo[1,5-a]pyridines has been achieved either via a regioselective [3 + 2] cycloaddition of N-aminopyridines with alkynes or by thermal cyclization of disubstituted azirines. Subsequent palladium-catalyzed introduction of pyridines or de novo synthesis of pyrimidines affords inhibitors of p38 kinase.