The role of subcellular localization in assessing the cytotoxicity of iodine-125 labeled iododeoxyuridine,iodotamoxifen, and iodoantipyrine

There is abundant evidence that Auger effects from¹²⁵I are singularly damaging if localized within DNA as the thymidine analogue¹²⁵I-iododeoxyuridine (¹²⁵IUdR). Recent work with¹²⁵I-labeled intercalating agents and steroid sex hormones extends these observations by showing cytotoxicity with¹²⁵I in sites other than the DNA backbone. We have compared the cytotoxicity of¹²⁵IUdR,¹²⁵I-iodotamoxifen, a non-steroidal antiestrogen that is translocated from the cytoplasm to the nucleus of receptor containing cells, and¹²⁵I-iodoantipyrine, a biological indicator of the body water space, in human breast cancer cells (MCF-7) and report that cytotoxicity is critically dependent upon subcellular localization. When clonogenic survival of MCF-7 cells is expressed as a function of the concentration of¹²⁵IUdR,¹²⁵ITAM and¹²⁵IAP in the culture media, the D₃₇ values are 8·10⁻⁴, 2.3 and 68 μCi/ml, respectively. However, when survival is expressed as a function of the nucleic acid and protein subcellular fraction,¹²⁵ITAM is just about as toxic as¹²⁵IUdR localized within the DNA backbone.     

Inter- and intraspecific variation in excited-state triplet energy transfer rates in reaction centers of photosynthetic bacteria

In protein-cofactor reaction center (RC) complexes of purple photosynthetic bacteria, the major role of the bound carotenoid (C) is to quench the triplet state formed on the primary electron donor (P) before its sensitization of the excited singlet state of molecular oxygen from its ground triplet state. This triplet energy is transferred from P to C via the bacteriochlorophyll monomer B(B). Using time-resolved electron paramagnetic resonance (TREPR), we have examined the temperature dependence of the rates of this triplet energy transfer reaction in the RC of three wild-type species of purple nonsulfur bacteria. Species-specific differences in the rate of transfer were observed. Wild-type Rhodobacter capsulatus RCs were less efficient at the triplet transfer reaction than Rhodobacter sphaeroides RCs, but were more efficient than Rhodospirillum rubrum RCs. In addition, RCs from three mutant strains of R. capsulatus carrying substitutions of amino acids near P and B(B) were examined. Two of the mutant RCs showed decreased triplet transfer rates compared with wild-type RCs, whereas one of the mutant RCs demonstrated a slight increase in triplet transfer rate at low temperatures. The results show that site-specific changes within the RC of R. capsulatus can mimic interspecies differences in the rates of triplet energy transfer. This application of TREPR was instrumental in defining critical energetic and coupling factors that dictate the efficiency of this photoprotective process.     

The 1.4 and 248 nm radiation response of chemically amplified resists containing arylmethyl sulfone photoacid generators

The X-ray (1.4 nm) and deep UV (248 nm) radiation responses of chemically amplified photoresists incorporating arylmethyl sulfone photoacid generators were evaluated. The arylmethyl sulfones were primarily derivatives of benzyl phenyl sulfone, selected to reveal the importance of desulfonylation and internal abstraction with regard to the photochemical efficiency of acid generation. At 1.4 nm, benzyl phenyl sulfone gave a much more sensitive resist than dibenzyl sulfone, while the methyl derivatives of benzyl phenyl sulfone did not give much improvement over the parent compound. This suggests that desulfonylation is more important than internal abstraction for increased photochemical efficiency. At 248 nm, similar trends were observed, but with some modifications arising from the variation in extinction coefficient among the sulfones. Sensitivities at or below 20 mJ/cm² were obtained for both wavelength ranges.     

Site-specific incorporation of the mucin-type N-acetylgalactosamine-alpha-O-threonine into protein in Escherichia coli

Glycosylation is a prevalent posttranslational process capable of augmenting and modulating protein function. Efficient synthesis of high-purity, homogeneous glycoproteins is essential for the study of unique protein glycoforms and for the manufacture of therapeutically relevant forms. A promising new strategy for controlled in vivo synthesis of glycoproteins was recently established using suppressor tRNA technology. Using an evolved tRNA aminoacyl synthetase-tRNA pair from Methanococcus jannaschii, the glycosyl amino acid, N-acetylglucosamine-beta-O-serine (GlcNAc-beta-Ser), was site-specifically introduced into proteins cotranslationally in Escherichia coli. Herein, we report the evolution of a new tRNA aminoacyl synthetase-tRNA pair that has expanded the repertoire of glycoproteins that can be expressed in E. coli to contain the other major O-linked glycan, N-acetylgalactosamine-alpha-O-threonine (GalNAc-a-Thr).     

Studies on the biosynthesis of secobotryane skeleton

The labelling and coupling patterns of secobotrytrienediol, biosynthesised from [1-¹³C] and [1,2-¹³C₂]-acetate by the fungus Botrytis cinerea, have been used to define the mode of formation and the biogenetic origin of secobotrytrienediol. [10-²H]-Botrydiol was not incorporated into the secobotryane skeleton. In addition, this feeding experiment led to the isolation of three new unlabelled derivatives possessing a secobotryane skeleton, secobotrydiene-3,10,15-triol, secobotrydiene-3,4,10,15-tetraol, and secobotrytriene-10,12,15-triol.     

The synthesis of P-chiral amino acid-derived phosphonamidic anhydrides

The synthesis of an array of P-chiral amino acid-derived phosphonamidic anhydrides is described. These anhydrides are prepared by condensation of allylated amino acids 19-22 with methyl- or vinylphosphonic dichlorides 23 or 24 to produce three diastereomeric anhydrides 4-11a-c in good to excellent yields. The mechanistic issues concerning anhydride formation are discussed and supported by experimental results. Vinylphosphonamidic anhydrides 8-11 are further derivatized via the ring-closing metathesis (RCM) reaction to yield amino acid-derived bicyclic phosphonamidic anhydrides.     

Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 x 10(-3) mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm(-2)). Approximately 10(-4) mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.     

High-temperature measurements of the reactions of OH with toluene and acetone

The reaction of hydroxyl [OH] radicals with toluene [C6H5CH3] was studied at temperatures between 911 and 1389 K behind reflected shock waves at pressures of approximately 2.25 atm. OH radicals were generated by rapid thermal decomposition of shock-heated tert-butyl hydroperoxide [(CH3)3-CO-OH], and monitored by narrow-line width ring dye laser absorption of the well-characterized R1(5) line of the OH A-X (0,0) band near 306.7 nm. OH time histories were modeled by using a comprehensive toluene oxidation mechanism. Rate constants for the reaction of C6H5CH3 with OH were extracted by matching modeled and measured OH concentration time histories in the reflected shock region. Detailed error analyses yielded an uncertainty estimate of +/-30% at 1115 K for the rate coefficient of this reaction. The current high-temperature data were fit with the lower temperature measurements of Tully et al. [J. Phys. Chem. 1981, 85, 2262-2269] to the following two-parameter form, applicable over 570-1389 K: k3 = (1.62 x 10(13)) exp(-1394/T [K]) [cm3 mol(-1) s(-1)]. The reaction between OH radicals and acetone [CH3COCH3] was one of the secondary reactions encountered in the toluene + OH experiments. Direct high-temperature measurements of this reaction were carried out at temperatures ranging from 982 to 1300 K in reflected shock wave experiments at an average total pressure of 1.65 atm. Uncertainty limits were estimated to be +/-25% at 1159 K. A two-parameter fit of the current data yields the following rate expression: k6 = (2.95 x 10(13)) exp(-2297/T [K]) [cm3 mol(-1) s(-1)].     

Attractor vicinity decay for a cellular automaton

The temporal decay of an attractor's vicinity for a domain-wall dominated cellular automaton (CA) is studied. Using selected initial pattern ensembles, state space structures in this high-dimensional nonlinear spatial system can be identified via the resulting decay to its attractors. Considered over a range of lattice sizes, the decay behavior falls into three main classes, each of which shows a characteristic profile. The first consists of even-size lattices showing a decelerating decay to small nonattracted ensemble fractions. The second class, also for even lattices, is a catastrophic decay to very small or vanishing nonattracted fractions. The third class also shows catastrophic decay and contains all odd-size lattices. Stochastic models are constructed that mimic the behavior of typical lattices throughout their evolution until finite-size effects appear. Weak additive noise causes all states on all lattices to fall into the attractor. In the end we find it overwhelmingly likely that the recently proposed attractor-basin portrait captures the CA's qualitative dynamics.