BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Femtosecond study of Cu(H(2)O) dynamics

The short-time nuclear dynamics of Cu(H(2)O) is investigated using femtosecond photodetachment-photoionization spectroscopy and time-dependent quantum wave packet calculations. The Cu(H(2)O) dynamics is initiated in the electronic ground state of the complex by electron photodetachment from the Cu(-)(H(2)O) complex, where hydrogen atoms are oriented toward Cu. Several time-resolved resonant multiphoton ionization schemes are used to probe the ensuing reorientation and dissociation. Immediately following photodetachment, the neutral complex is far from its minimum energy geometry and possesses an internal energy comparable to the Cu-H(2)O dissociation energy and undergoes both large-amplitude H(2)O motion and dissociation. Dissociation is observed to occur on three distinct time scales: 0.6, 8, and 100 ps. These results are compared to the results of time-dependent J=0 wave packet calculations, propagating the initial anion vibrational wave functions on the ground-state potential of the neutral complex. An excellent agreement is obtained between the experimental results and the ionization signals derived from the calculated probability amplitudes. Related experiments and calculations are carried out on the Cu(D(2)O) complex, with results very similar to those of Cu(H(2)O).     

Radiometric and fluorimetric determination of aminosilanes and protein covalently bound to thermally pretreated glass substrates

Derivatization of solid soda-lime glass spheres with aminosilanes and the stability of these groups near physiological pH in flow streams of aqueous buffered solutions are described. The presence of immobilized and adsorbed amines in the nanomolar range is confirmed by using two independent methods, one based on a radiotracer and the other on the fluorescent marker, fluorescamine. A method for covalently attaching bovine serum albumin to the beads is described.     

Synthesis and H-NMR complexation studies of alkalimetal bithioxanthylidene crown ether complexes

Two synthetic routes to a novel class of sterically overcrowded alkenes, bithioxanthylidene crown ethers 1, are described. The ¹H spin-lattice relaxation times (T₁) of the crown ethers as well as those of the corresponding complexes with Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺ in CDCl₃ were measured and the results were interpreted in terms of complexation affinities.     

Wittig and horner-wittig coupling reactions of 2-substituted cyclic ethers and their application to spiroketal synthesis

Wittig and Horner-Wittig coupling reactions of tetrahydropyran or tetrahydrofuran 2-triphenylphosphonium salts or 2-diphenylphosphine oxides with aldehydes and lactols affords good yields of the corresponding enol ethers. In selected examples these enol ether products may be further converted to spiroketals some of which are natural pheromones derived from $\text{Dacus oleae}$ and $\text{Paravespula vulgaris}$.     

Origin of enantioselection in chiral alcohol oxidation catalyzed by Pd[(-)-sparteine]Cl2

A kinetic investigation into the origin of enantioselectivity for the Pd[(-)-sparteine]Cl(2)-catalyzed aerobic oxidative kinetic resolution (OKR) is reported. A mechanism to account for a newly discovered chloride dissociation from Pd[(-)-sparteine]Cl(2) prior to alcohol binding is proposed. The mechanism includes (1) chloride dissociation from Pd[(-)-sparteine]Cl(2) to form cationic Pd(-)-sparteine]Cl, (2) alcohol binding, (3) deprotonation of Pd-bound alcohol to form a Pd-alkoxide, and (4) beta-hydride elimination of Pd-alkoxide to form ketone product and a Pd-hydride. Utilizing the addition of (-)-sparteine HCl to control the [Cl(-)] and [H(+)] and the resulting derived rate law, the key microscopic kinetic and thermodynamic constants were extracted for each enantiomer of sec-phenethyl alcohol. These constants allow for the successful simulation of the oxidation rate in the presence of exogenous (-)-sparteine HCl. A rate law for oxidation of the racemic alcohol was derived that allows for the successful prediction of the experimentally measured k(rel) values when using the extracted constants. Besides a factor of 10 difference between the relative rates of beta-hydride elimination for the enantiomers, the main enhancement in enantiodetermination results from a concentration effect of (-)-sparteine HCl and the relative rates of reprotonation of the diastereomeric Pd-alkoxides.     

Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy

The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.     

DEMONSTRATION THAT PHOSPHORESCENT 6-BROMO-2-NAPHTHYL SULFATE CAN BE USED TO PROBE HEME ACCESSIBILITY IN HEME PROTEINS

The phosphorescence properties of 6-bromo-2-naphthyl sulfate (BNS) in aqueous solution were studied. The phosphorescence lifetime is several hundred microseconds and is self-quenched. Although a fluorescent photoproduct is formed from BNS, it does not interfere with the decay properties of triplet-state BNS and its utility as a probe of the accessibility of the heme group in heme proteins. Quenching of BNS phosphorescence does not occur for the non-heme protein lysozyme and apomyoglobin but occurs by a dynamic mechanism with a quenching constant of 1-2 x 10(9) M-1 s-1 for cytochrome c and myoglobin and with a quenching constant of 6.2 x 10(9) M-1 s-1 for protoporphyrin IX. The phosphorescence of an inclusion complex of 1-bromonaphthalene and beta-cyclodextrin is not quenched by heme-containing proteins. The temperature and viscosity dependencies of the rate with which BNS phosphorescence is quenched by microperoxidase-11 are consistent with unit quenching efficiency. These results indicate that quenching of BNS phosphorescence occurs only upon contact with the quencher, and the quenching constant can be used to assess the degree of accessibility of the heme group.     

A general strategy for the diastereoselective synthesis of 2,6-diaryl-3,7-dioxabicyclo[3.3.0]octane lignans

A strategy for the stereoselective synthesis of all the possible diastereoisomers of the 2,6-diaryl-3,7-dioxabicyclo[3.3.0]octane (furofuran) lignans from a single dihydrofuran precursor is described. The key steps involve a diastereocontrolled templated cationic cyclization followed by stereoselective reduction of the resulting methyl glycoside.     

Reactivity of DNA guanyl radicals with phenolate anions

Guanine bases are the most easily oxidized sites in DNA. Electron-deficient guanine species are major intermediates produced in DNA by the direct effect of ionizing radiation (ionization of the DNA itself) because of preferential hole migration within DNA to guanine bases. By using thiocyanate ions to modify the indirect effect (ionization of the solvent), we are able to produce these single-electron-oxidized guanine radical species in dilute aqueous solutions of plasmid DNA where the direct effect is negligible. The guanyl radical species produce stable modified guanine products. They can be detected in the plasmid by converting them to strand breaks after incubation with a DNA repair enzyme. If a phenol is present during irradiation, the yield of modified guanines is decreased. The mechanism is reduction of the guanine radical species by the phenol. It is possible to derive a rate constant for the reaction of the phenol with the guanyl radical. The pH dependence shows that phenolate anions are more reactive than their conjugate acids, although the difference for guanyl radicals is smaller than with other single-electron-oxidizing agents. At physiological pH values, the reduction of a guanyl radical entails the transfer of a proton in addition to the electron. The relatively small dependence of the rate constant on the driving force implies that the electron cannot be transferred before the proton. These results emphasize the potential importance of acidic tyrosine residues and the intimate involvement of protons in DNA repair.