Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.

Comparative study of the radiation-induced grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) and polypropylene substrates. I: Kinetics and structural investigation

A comparative study has been made of the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropyl vinyl ether) (PFA) and polypropylene (PP) substrates, using the simultaneous irradiation method. Effects of grafting conditions such as monomer concentrations, type of solvent, dose rate and irradiation dose on the grafting yield were investigated. Under the same grafting conditions it was found that a higher degree of grafting of styrene was obtained using a mixture of dichloromethane/methanol solvents for PFA and methanol for PP and the degree of grafting was higher in PP than in PFA at all doses. However, the micro-Raman spectroscopy analysis of the graft revealed that, for the same degree of grafting, the penetration depth of the grafted polystyrene into the substrate was higher in PFA than in PP substrates. In both polymers the crystallinity was hardly affected by the grafting process and the degree of crystallinity decreased slightly with grafting dose. The dependence of the initial rate of grafting on the dose rate and the monomer concentration was found to be 0.6 and 1.4 order for PFA and 0.15 and 2.2 for PP, respectively. The degree of grafting increased with increasing radiation dose in both polymers. However, the grafting yield decreased with an increase in the dose rate. The increase in the overall grafting yield for PFA and PP was accompanied by a proportional increase in the penetration depth of the graft into the substrates. (C) 2003 Society of Chemical Industry.

Radical formation on gamma-radiolysis of poly [(methacrylic acid)-co-acrylonitri

The radicals formed on gamma-radiolysis of a series of copolymers of methacrylic acid and acrylonitrile have been investigated by ESR spectroscopy. This series of copolymers spanned the full composition range and the study was carried out at 77 K and ambient temperature. The radicals formed in the copolymers at 77 and 303 K were found to be similar to those found in the two homopolymers, but in the intermediate composition range the presence of acrylonitrile propagation radicals was also detected. These radicals were not observed to be formed in significant quantities on the radiolysis of polyacrylonitrile. They are believed to result from a scission of the main chain at methacrylic acid/acrylonitrile diad sequences following loss of the methacrylic acid carboxyl group. At 77 K, the copolymers with high methacrylic acid contents were found to be more sensitive to radical formation than the methacrylic acid homopolymer, but this enhanced sensitivity was not evident at ambient temperature, where the G-values for radical formation for the copolymers were slightly less than the values for the homopolymers. (C) 2003 Society of Chemical Industry.

A study of the radiation degradation of styrene/alkane and α-methylstyrene/alkane copolymers

The effects of copolymer composition and microstructure on the radiation chemistry of styrene/alkane and alpha-methylstyrene/alkane copolymers have been studied. The primary radical species formed on radiolysis of the copolymers at 77 K, and identified by ESR spectroscopy, are the same as those formed during radiolysis of the homopolymers. The yields of radicals for the copolymer are as predicted assuming that the cross-section is proportional to the electron density of each component; however, there is some evidence of radical migration to aromatic groups at 77 K. Changes in molecular structure on irradiation were detected by using C-13 NMR spectroscopy. Evidence of the consumption of terminal double bonds, and chain scission in alpha-methylstyrene/alkane copolymers was found. Measurements of viscosity supported the mechanism of cross-linking predominating in styrene/alkane copolymers, while in alpha-methylstyrene/alkane copolymers chain scission was the major result of irradiation. (C) 2003 Society of Chemical Industry.

A study of the radiation chemistry of poly(chlorotrifluoroethylene) by ESR spectroscopy

The ESR spectra of poly(chlorotrifluoroethylene) were recorded following gamma-radiolysis under vacuum at room temperature and 77 K. The very broad spectrum at 77 K revealed little fine structure with which to identity the radicals formed upon irradiation, but subsequent photobleaching and annealing studies, together with radiolytic studies at higher temperatures, afforded scope for making radical assignments. Both main-chain radicals and a range of chain-end radicals have been identified. The G-values for radical formation were 1.55, 0.36 and 0.32 at 77 K, 273 K and room temperature, respectively. (C) 2003 Elsevier Science Ltd. All rights reserved.

High energy radiation grafting of fluoropolymers

Fluoropolymers are known as chemically inert materials with good high temperature resistance, so they are often the materials of choice for harsh chemical environments. These properties arise because the carbon-fluorine bond is the strongest of all bonds between other elements and carbon, and, because of their large size, fluorine atoms can protect the carbon backbone of polymers such as poly(tetrafluoroethylene), PTFE, from chemical attack. However, while the carbon-fluorine bond is much stronger than the carbon hydrogen bond, the G values for radical formation on high energy radiolysis of fluoropolymers are roughly comparable to those of their protonated counterparts. Thus, efficient high energy radiation grafting of fluoropolymers is practical, and this process can be used to modify either the surface or bulk properties of a fluoropolymer. Indeed, radiation grafted fluoropolymers are currently being used as separation membranes for fuel cells, hydrophilic filtration membranes and matrix substrate materials for use in combinatorial chemistry. Herein we present a review of recent studies of the high energy radiation grafting of fluoropolymers and of the analytical methods available to characterize the grafts. (C) 2003 Elsevier Ltd. All rights reserved.

Sensitivity of Helicoverpa armigera nucleopolyhedrovirus polyhedra to sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is commonly used to extract polyhedra from infected cells and diseased dead larval tissues. It was found, however, that 80% of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) polyhedra produced via cell culture were damaged after 30 min of 0.5% SDS treatment whereas only 20% of in vivo produced polyhedra were damaged by the same treatment. Transmission and scanning electron microscopy revealed that the damaged polyhedra had lost their polyhedron envelopes and virions were dislodged from the polyhedrin matrix, leaving empty spaces that were previously occupied by the occluded virions. Up to 20% in vitro produced polyhedra were resistant to SDS and remained intact, even after a 24 h exposure to SDS. This sensitivity to SDS was observed across a range of cell culture media, including serum supplemented media. Electron microscopy also revealed that the inferior polyhedron envelope of in vitro produced polyhedra is likely due to poor interaction between the polyhedron envelope, polyhedron envelope protein (PEP), and polyhedrin matrix. The PEP gene was cloned and sequenced and mutations in this gene were ruled out as an explanation. In vitro produced polyhedra that were passed through insect larva once were resistant to SDS, indicating that a critical component is lacking in insect cell culture medium used for producing HaSNPV or the cells growing in culture are inefficient in some ways in relation to production of polyhedra. (C) 2002 Elsevier Science (USA). All rights reserved.