Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells

Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.     

Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide–nanoparticle conjugates

Novel methods for application of oligonucleotide–gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide–gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for F 508, M470V, R74W and R75Q mutations.     

Mechanism of Exciplex Decay: The Quantum Yields and the Rate Constants of Triplet Formation from 9-Cyanophenanthrene Exciplexes

The quantum yields of the formation of triplet states from the exciplexes of 9-cyanophenanthrene with 1,2,3- and 1,3,5-trimethoxybenzenes in solvents of different polarity and the rate constants of intersystem crossing in these exciplexes were measured. In the solvents of weak and intermediate polarity, the exciplexes decay predominantly via internal conversion and intersystem crossing, whereas the decay in polar solvents occurs additionally via dissociation into radical ions.     

Discovery of a potent,non-peptide bradykinin B1 receptor antagonist

Bradykinin (BK) plays an important role in the pathophysiological processes accompanying pain and inflammation. Selective bradykinin B1 receptor antagonists have been shown to be anti-nociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. We have explored chemical modifications in a series of dihydroquinoxalinone sulfonamides to evaluate the effects of various structural changes on biological activity. The optimization of a screening lead compound, facilitated by a homology model of the BK B1 receptor, culminated in the discovery of a potent human BK B1 receptor antagonist. Results from site-directed mutagenesis studies and experiments in an animal pain model are presented.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic-mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-Sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using 18O-labelled amino acids and gas chromatography-mass spectrometry.     

Direct observation of enantiomer discrimination of epoxides by chiral salen complexes using ENDOR

Electron nuclear double resonance (ENDOR) spectroscopy was used to investigate the weak enantioselective binding between chiral salen complexes [VO(1)] ((R,R)- and (S,S)-vanadyl N,N'-bis(3,5-di-tert-butylsalcylidene)-1,2-cyclohexanediamine) and chiral epoxides (e.g., (R)-/(S)-propylene epoxide, 5) in frozen (10 K) solution. Differences in epoxide binding by enatiomers of [VO(1)] was evidenced by changes to the 1H epoxide derived peaks in the ENDOR spectra, such that (R,R)-[VO(1)] + (R)-5 and (R,R)-[VO(1)] + (S)-5 yield noticeably different spectra. These changes were assigned to the small structural differences between the diastereomeric metal-epoxide adducts. Simulation of the spectra revealed differences in the VO...1Hepoxide distances for the diastereomeric pairs, which was confirmed by a complementary set of density functional theory (DFT) calculations. While the epoxide molecule is very weakly coordinated, ENDOR measurements of the racemic complex in racemic epoxide nevertheless indicated the preferential coordination of the (R)-5 to (R,R)-[VO(1)] (likewise (S)-(5) to (S,S)-[VO(1)]), which is favored over the binding of (S)-5 epoxide to (R,R)-[VO(1)] (and likewise (R)-5 epoxide to (S,S)-[VO(1)]). This demonstrates the unique power of the ENDOR technique to resolve weak chiral interactions for which EPR spectroscopy alone lacks sufficient resolution.     

Electron donor and acceptor properties of alkyl substituents

The polar effects of alkyl substituents in electrophilic and nucleophilic chemical and electronic transitions is discussed. The question of the importance of hyperconjugation in the electron donor properties of alkyl substituents is raised. In view of the cogent arguments of Dewar, it is doubtful whether quantum mechanical calculations embodying hyperconjugation constitute proof of this effect. That the art of quantum mechanics may not yet be sufficiently developed to be used as proof for or against secondary resonance effects also is evidenced by the calculations of Simpson, who found that an internal dispersion force model (in which conjugation was neglected) reproduced the properties of butadiene just as satisfactorily as the models embodying conjugation. The experimental facts do not unequivocally support the hyperconjugation hypothesis and indeed are, at least in part, contradictory to it. In particular, the demonstration that the Baker-Nathan Effect² may be due to the influence of alkyl substituents on the differential solvation of ground and transition states casts doubt on the interpretation that this experimental effect is due to a dominant role of C-H hyperconjugation.

In nucleophilic chemical reactions, rate or equilibrium constants for para (or meta) alkyl derivatives are somewhat smaller than those of the corresponding hydrogen compounds. A number of authors have interpreted this in terms of a permanent electron donor role of alkyl substituents (e.g. by hyperconjugation) relative to the hydrogen substituent. However, this static viewpoint of substituent effects fails to account for the finding that p-alkyl substituents function as apparent electron acceptors (relative to the p-hydrogen substituent) in appreciably lowering the energy of the nucleophilic principal electronic transition of phenol, anisole, aniline and N,N-dimethylaniline. These results are qualitatively rationalized in terms of ‘substituent-polarizability” and electronegativity.

The p-neopentyl substituent lowers the energy of both electrophilic and nucleophilic electronic transitions to an appreciably greater extent than either the p-methyl or p-t-butyl substituent. This extra stabilizing effect of the neopentyl substituent on both electron deficient and electron rich centers may be due to an internal dispersion force interaction, since the geometry of the neopentyl compounds is particularly favorable for such an interaction.     

Dose response and post-irradiation characteristics of the Sunna 535-nm photo-fluorescent film dosimeter

Results of characterization studies on one of the first versions of the Sunna photo-fluorescent dosimeter™ have previously been reported, and the performance of the red fluorescence component described. This present paper describes dose response and post-irradiation characteristics of the green fluorescence component from the same dosimeter film (Sunna Model γ), which is manufactured using the injection molding technique. This production method may supply batch sizes on the order of 1 million dosimeter film elements while maintaining a signal precision (1σ) on the order of ±1% without the need to correct for variability of film thickness. The dosimeter is a 1 cm×3 cm polymeric film of 0.5-mm thickness that emits green fluorescence at intensities increasing almost linearly with dose. The data presented include dose response, post-irradiation growth, heat treatment, dosimeter aging, dose rate dependence, energy dependence, dose fractionation, variation of response within a batch, and the stability of the fluorimeter response. The results indicate that, as a routine dosimeter, the green signal provides a broad range of response at food irradiation (0.3–5 kGy), medical sterilization (5–40 kGy), and polymer cross-linking (40–250 kGy) dose levels.     

Negative ion electrospray tandem mass spectrometric structural characterization of leukotriene B4 (LTB4) and LTB4-derived metabolites

The low energy collision induced dissociation (CID) of the carboxylate anions generated by electrospray ionization of leukotriene B₄ (LTB₄) and 16 of its metabolites was studied in a tandem quadrupole mass spectrometer. LTB₄ is a biologically active lipid mediator whose activity is terminated by metabolism into a wide variety of structural variants. The collision-induced dissociation spectra of the carboxylate anions revealed structurally informative ions whose formation was determined by the position of hydroxyl substituents and double bonds present in the LTB₄ metabolite. Major ions resulted from charge remote α-hydroxy fragmentation or charge directed α-hydroxy fragmentation. The conjugated triene moiety present in some metabolites was proposed to undergo cyclization to a 1,3-cyclohexadiene structure prior to charge remote or charge driven a-hydroxy fragmentation. The mechanisms responsible for all major ions observed in the CID spectra were studied using stable isotope labeled analogs of the LTB₄ metabolites. In general, the collision-induced decomposition of carboxylate anions produced unique spectra for all LTB₄ derived metabolites. The observed decomposition product ions from the carboxylate anion could be useful in developing assays for these molecules in biological fluids.     

Multiple forms of formamidopyrimidine-DNA glycosylase produced by alternative splicing in Arabidopsis thaliana

Formamidopyrimidine-DNA glycosylase (FPG) catalyzes the initial steps in the repair of DNA containing oxidized purines. Two cDNA clones from Arabidopsis thaliana encoding homologs of bacterial FPG have previously been described. We now report that there are at least five additional variants of FPG mRNA in Arabidopsis, each apparently produced from the same gene (AtMMH) by alternative splicing. Thus, AtMMH, like at least four other genes in the base excision repair pathway of human cells, produces multiple forms of protein product through alternative splicing. The variant forms of Arabidopsis FPG may be localized in different locations in the cells, may have different preferences for oxidized substrates, and/or may recruit different proteins that guide the subsequent steps of base excision repair.