Susceptibility of the trauma symptom inventory to malingering.

This study examined the sensitivity and specificity of the Trauma Symptom Inventory (TSI; Briere, 1995), a self-report measure of psychological sequelae of potentially traumatic events, to malingering. An optimal cutting score for a validity scale--Atypical Responding (ATR)--designed to identify exaggeration or other unusual response sets was developed in an analogue sample of 155 college students and subsequently applied to TSI profiles from several samples of patients with various psychiatric disorders. Use of a cross-validated T-score cutoff of 61 and below on the ATR scale produced good sensitivity (81%) and specificity (92%) rates in the analogue sample. Participants in the analogue sample who reported a history of traumatic experiences were no more able to successfully malinger trauma symptoms than were participants without such histories. Furthermore, false-positive rates in the clinical samples were generally low, suggesting that relatively few genuinely symptomatic individuals would be misclassified as malingering.

Solution structures of psoralen monoadducted and cross-linked DNA oligomers by NMR spectroscopy and restrained molecular dynamics.

We have used two-dimensional 1H NMR spectroscopy to determine the solution structures of the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furanside monoadducted (MAf) and the photoisomeric HMT interstrand cross-linked (XL) DNA oligonucleotide d(5'-GCGTACGC-3')2. The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for both the MAf and XL from both A-form and B-form dsDNA starting structures. The root-mean-square (rms) deviations of the coordinates for the 40 structures for the MAf and XL were 1.12 and 1.10 A, respectively. The rmsd of the MAf with respect to the XL is 2.20 A. The local DNA structure is distorted in both adducts, with the helix unwound by 34 degrees and 25 degrees for the MAf and XL, respectively, and an overall helical repeat of 11 base pairs, caused by intercalation of the HMT. The MAf is a photochemical intermediate on the path to interstrand XL. Considerable local structural distortion is induced by both adducts, but the DNA returns to B-form structure within three base pairs of the damage site. There is no significant bend in the helix axis of either the MAf or the XL. We have evaluated the accuracy of the two major methods of converting NOESY data into interproton distances, the isolated spin-pair approximation (ISPA) and the complete relaxation rate matrix analysis (RMA). Both methods were evaluated by comparing the resulting calculated interproton distances generated to known covalently fixed distances in the HMT. The overall structures were evaluated by checking their agreement with biophysical evidence from non-NMR techniques. Only the modified RMA method gave correct interproton distances.

The structure of U17 isolated from Streptomyces clavuligerus and its properties as an antioxidant thiol.

The predominant low-molecular-mass thiol produced by streptomycetes is a cysteine derivative previously designated as U17 [Newton, G. L., Fahey, R. C., Cohen, G. & Aharonowitz, Y. (1993) J. Bacteriol. 175, 2734-2742]. In this study we report the elucidation of the structure of the monobromobimane derivative of U17, which establishes the structure of U17 as 2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl-myo-inositol. The presence of the N-acetylcysteine moiety was indicated by formation of N-acetylcysteine-monobromobimane during acid hydrolysis of the monobromobimane derivative of U17. Complete hydrolysis released 1 mol glucosamine/mol cysteine as determined by carbohydrate and amino acid analysis. High-resolution mass spectral analysis gave a precise mass consistent with the molecular formula C27H40N4O14S. Analysis of 13C-NMR, one-dimensional 1H-NMR and two-dimensional NMR experiments identified the remaining C6H12O6 moiety as myo-inositol, confirmed the presence of N-acetylcysteine and glucosamine, and established the connectivity of the components. Two chemical properties of this novel thiol make it suitable as an intracellular storage form of cysteine and as an antioxidant thiol. First, it undergoes heavy-metal-ion catalyzed autoxidation at a rate dramatically lower than that for cysteine and markedly lower than that for glutathione or N-acetylcysteine. Secondly, the alpha-(1-->1) glycosidic link between glucosamine and myo-inositol is resistant to acid hydrolysis, hydrolysing at a rate comparable to that of the two amide bonds in the molecule.

DNA structural reorganization upon conversion of a psoralen furan-side monoadduct to an interstrand cross-link: implications for DNA repair.

We have used 1H NMR spectroscopy to determine the structural changes induced in the DNA oligomer d(5'-GCGTACGC-3')2 upon conversion of the 4'-hydroxy-methyl-4,5',8-trimethylpsoralen-DNA furan-side monoadduct (MAf) to the interstrand cross-link (XL). The MAf is a photochemical intermediate on the path to interstrand XL and has the psoralen intercalated into the helix. The local DNA structure is distorted in both adducts, but it returns to normal within three base pairs. The formation of XL requires displacement of the psoralen toward the initially unmodified strand, accompanied by a change in the hybridization of the thymine C-5 and C-6 carbons and a change in the local helix twist. The MAf is intercalated in the helix. There is no significant bend in the helix axis of either the MAf or XL. There are significant changes in the local helix dynamics upon photoadduct formation that may be recognized by cellular DNA repair enzyme systems. We hypothesize that the repair enzymes target lesions by detecting the conformational flexibility of the sugar-phosphate backbone induced by DNA-damaging agents.

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure.

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.

Antiparallel side-by-side dimeric motif for sequence-specific recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin.

The designed peptide 1-methylimidazole-2-carboxamide netropsin (2-ImN) binds specifically to the sequence 5'-TGACT-3'. Direct evidence from NMR spectroscopy is presented that this synthetic ligand binds DNA as a 2:1 complex, which reveals that the structure is an antiparallel dimer in the minor groove of DNA. This is in contrast to the 1:1 complexes usually seen with most crescent-shaped minor groove binding molecules targeted toward A+T-rich tracts but reminiscent of a dimeric motif found for distamycin at high concentrations. These results suggest that sequence-dependent groove width may play an important role in allowing an expanded set of DNA binding motifs for synthetic peptides.

Determination of the acid dissociation constants for WR-1065 by proton NMR spectroscopy.

The acid dissociation constants for N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were determined in D2O by 360- and 500-MHz NMR spectroscopy. Results obtained at 0.21 M initial ionic strength and 26 degrees C were corrected to 25 degrees C yielding pKD1 = 8.28 +/- 0.04, pKD2 = 9.88 +/- 0.07, and pKD3 = 11.58 +/- 0.03. Correction of these values for the effect of the deuterium isotope upon the ionization reaction yielded dissociation constants in water of pKH1 = 7.69 +/- 0.09, pKH2 = 9.35 +/- 0.09, and pKH3 = 11.10 +/- 0.08. Analysis of the changes in chemical shift with pD indicated that the first ionization occurs largely through ionization of the thiol group (approximately 67%) and to a lesser extent the secondary ammonium group (approximately 30%), whereas the third ionization involves mainly the secondary ammonium group (approximately 65%) and to a lesser extent the primary ammonium group (approximately 30%). Estimates of the microscopic pK values for WR-1065 were also obtained from the results.

Recent advances in the synthesis and structure determination of site specifically psoralen-modified DNA oligonucleotides.

We have developed novel methods for the preparation of multimicromole quantities of extremely pure, uniquely photoadducted psoralen-DNA cross-links, furan-side monoadducted DNA and pyrone-side monoadducts. Psoralen cross-linked and furan-side monoadducted DNA were produced by employing high intensity argon ion and krypton ion lasers as light sources. Pyrone-side monoadducts were prepared by base-catalyzed photoreversal of psoralen cross-links. The various psoralen-adducted DNA oligomers were efficiently purified by high performance liquid chromatography. These methods have permitted us to synthesize 4 mumol each of a self-complementary 8-mer d(GCGTACGC) 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furan-side monoadduct and HMT cross-link. Preliminary nuclear magnetic resonance (NMR) data on the HMT cross-linked 8-mer d(GCGTACGC) have been obtained which confirmed the presence of the diadducted psoralen at the unique 5'TpA3' site. NMR data obtained from the 8-mer furan-side monoadduct revealed that the psoralen molecule is intercalated into the DNA double helix. Preliminary crystals of 8-mer cross-linked DNA molecule have been grown. Conditions for the growth of X-ray diffraction-quality crystals and the further analysis of these crystals are now in progress.