IRRADIATION OF CIRCULAR DNA WITH 254 nm RADIATION OR SENSITIZATION IN THE PRESENCE OF Ag+ EVIDENCE FOR UNWINDING BY PHOTOPRODUCTS OTHER THAN PYRIMIDINE DIMERS

Abstract— Structural alterations of DNA irradiated with UV light were analyzed by the agarose gel technique. Relaxed, circular pAT 153 DNA molecules were sensitized by broad band radiation with a maximum at 313 nm in the presence of silver ions or irradiated with 254 nm light in buffer only. In both cases the electrophoretic mobility of DNA topoisomers was altered as a linear function of UV exposure. For DNA irradiated in the sensitized reaction the unwinding angle per site sensitive to Micrococcus luteus pyrimidine dimer endonuclease was found tobe–11.4°. This value is significantly smaller thanthe–14.3° already known for DNA topoisomers irradiated with 254 nm light. The irradiated DNAs were a very good substrate for the Escherichia coli photoreactivating enzyme (PRE). However, the photoenzymic removal of all sites sensitive to the endonuclease specific for pyrimidine dimers was not coupled to a full restoration of the original electrophoretic mobility. Thirty and 23% of the unwinding were still present in the photoreactivated topoisomers and the unwinding angles per pyrimidine dimer were then recalculatedas–10.1°and–8.7° for DNAs irradiated with 254 nm and sensitized, respectively. The limited difference between these two values could result from the different base composition of the pyrimidine dimers generated in the conditions of irradiation used. These results show that the tertiary structure of DNA is measureably altered by UV photodamages other than pyrimidine dimers.     

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.     

QUANTIFICATION OF PYRIMIDINE DIMERS AND APURINIC SITES IN DNAs OF UNIFORM LENGTH

Abstract— A new simple in vitro assay for the determination of pyrimidine dimers and/or apurinic/apyrimidinic sites in non-radioactive DNA has been developed. In this procedure, DNA substrates of uniform length-which may be supercoiled, partially relaxed, relaxed or linear-are treated with agents which produce specific single strand nicks at the site of the lesion. The number of lesions per molecule can be expressed as a function of the amount of single-stranded molecules left intact after the specific nicking treatment. Unreacted molecules, which retain the original uniform length, are separated from the other smaller reaction products by electrophoresis on an alkaline agarose gel. In the case of circular molecules, the substrate is linearized by the use of an appropriate restriction endonuclease before loading on the gel. The amount of intact DNA molecules is obtained by integrating the corresponding peak of absorption after densitometric scan of the negative of the gel picture. This assay can detect up to eight damaged sites per duplex molecule. This method could be particularly useful when dealing with mixtures of DNA with different degrees of supercoiling or for comparisons between linear and circular DNA substrates.     

Incandescent lamps can produce pyrimidine dimers in DNA

DNA molecules that have been exposed to light from a 150 W incandescent spot lamp are nicked by the Micrococcus luteus endonuclease specific for cyclobutyl-type pyrimidine dimers. The production of these enzyme-sensitive sites increases with increasing spot lamp exposure. These sites have been confirmed to be pyrimidine dimers by their property of being photoreversed by an E. coli photoreactivating enzyme. The emission spectrum of the lamp shows detectable output at wavelengths less than 320 nm. These results indicate that the sensitivity of the techniques utilized in this work can be used to detect low levels of contaminating UV radiation.     

Detection of Copper(II) in Water by Methylene Blue Derivatives

Two novel phenothiazin-5-ium derivatives bearing cyclam moieties ( 3 a⁺ and 3 b⁺ ) were synthesized and investigated as Cu²⁺ sensors. Both ligands show intense spectral changes in the presence of Cu²⁺ in aqueous solutions. The high molar extinction coefficient of the chromophore allows both naked eye detection and spectrophotometric quantitative determination of the cation at a micromolar-concentration scale. 3 a⁺ was found to outperform 3 b⁺ , showing negligible spectral changes in the presence of excesses of other metal cations.     

Electrochemically Driven Swinging of a Nitrobenzyl Pendant Arm in a Nickel Scorpionand Complex

A radical anion −NO₂^.− is formed upon an electrochemically reversible one-electron reduction of the square-planar Ni^II complex of N-nitrobenzylcyclam. The −NO₂^.− group goes to occupy an axial position of the metal ion, thus establishing a significant electronic interaction with the metal center. In particular, the ESR spectrum supports the occurrence of an electron transfer from −NO₂^.− to the metal, which therefore presents a significant Ni^I character. On re-oxidation, the nitrobenzyl side chain detaches and the Ni^II complex is restored, providing an example of a fully reversible redox driven intramolecular motion.     

Convenient TLC for the detection of a herbal ingredient in a food supplement

This study describes a practical and suitable approach to solve a real problem: the analysis of polyherbal products for which some specifications addressing the composition are lacking (e.g. chemical components). It is reported that an analytical procedure to verify the possible presence in a botanical supplement of a plant is not authorised for use in Italy. No reference of chemical standards for the known components of the plant is commercially available. For this reason and because a rapid response was required, more selective chromatographic techniques such as high-performance liquid chromatography, gas chromatography and liquid chromatography-mass spectrometry were excluded and thin layer chromatography was chosen for applying different experimental procedures. The original English label of the product, which is made by a leading US manufacturer and sold online by American and Italian distributors, lists the same herbal ingredients as the Italian label, but with an addition of Eurycoma longifolia Jack, which is not included in the list of plants admitted in supplements compiled by the Italian Ministry of Health. For greater certainty of the result, a high-performance thin layer chromatography method was then applied. On the basis of the results obtained, it was possible to confirm that E. longifolia Jack was not present in the product.     

A new equation for the thermal conductivity of organic compounds

This work presents a wide literature survey of the available data of the experimental thermal conductivity data of organic liquids. The experimental data were collected for 136 compounds belonging to the following families: refrigerants, alkanes, alkenes, aromatics, cycloalkanes, cycloalkenes, ethers, esters, ketones, carboxylic acids, and alcohols. The experimental data were regressed with the most reliable semi-empirical correlating methods existing in the literature and a reliable set of 4,584 experimental data was finally selected. The influence of several physical parameters on the thermal conductivity calculation is discussed and a new equation to represent the thermal conductivity of organic liquids at atmospheric pressure for temperatures below normal boiling point and at saturation for temperatures above the normal boiling point is presented. To minimize the deviation between the predictions and the experimental data and to find the optimal coefficients for the proposed equation, a statistical analysis was performed. The resulting equation is simple and is able to predict the thermal conductivities with low deviations for the major part of the collected data for the studied families.