Multiple conformations of an intercalated (-)-(7S,8R,9S, 10R)-N6-[10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence.

The structure of the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct at A7 of 5'-d(CGGACAAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N6-amino group of dA to (-)-(7S,8R,9R, 10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-DE2], was determined using molecular dynamics simulations restrained by 532 NOEs from 1H NMR. This was named the SRSR(61,3) adduct, derived from the N-rasprotooncogene at and adjacent to the nucleotides encoding amino acid 61 (underlined) of the p21 gene product. The solution structure of this adduct was best described as a mixture of two conformations in rapid equilibrium on the NMR time scale. The two populations differed in the pseudorotation angle of the sugar ring for the 5'-neighboring base A6, as determined from scalar coupling data. One population, estimated to be present at 53%, had the A6 deoxyribose in the C2'-endo conformation, while in the second conformation the A6 deoxyribose was in the C3'-endo conformation. NOEs between C5, A6, and SRSRA7 were either disrupted or weakened, as were those in the complementary strand between C15, T16, and T17. Major groove NOEs were observed between the benzo[a]pyrene aromatic protons, H1, H2, H3, H4, H5, and H6, and T16 CH3. Minor groove NOEs were observed between H1, H2, and H3 of benzo[a]pyrene and T16 H1' and H2' and T17 H1' and H2'. The benzo[a]pyrene protons H10, H11, and H12 showed NOEs to A6 H1', H2', and H2". The chemical shifts of the pyrenyl moiety were dispersed over a 1.9 ppm range. Upfield chemical shifts of 2.4 ppm for T16 N3H, 1.1 ppm for T17 N3H, 1.3 and 1.0 ppm for T16 H6 and CH3, 0.85 ppm for T16 H1', and 0.80 and 0.90 ppm for C15 H2' and H2" were observed. These observations were consistent with intercalation of the pyrenyl moiety toward the 5' direction of SRSRA7. The results were compared to the isomeric SRSR(61,2) adduct [I. S. Zegar, S. J. Kim, T. N. Johansen, P. J. Horton, C. M. Harris, T. M. Harris, and M. P. Stone (1996) Biochemistry 35, 6212-6224] and revealed the role of DNA sequence in modulating the conformation of this benzo[a]pyrene adduct.

Major groove (S)-alpha-(N6-adenyl)styrene oxide adducts in an oligodeoxynucleotide containing the human N-ras codon 61 sequence: conformations of the S(61,2) and S(61,3) sequence isomers from 1H NMR.

The (S)-alpha-(N6-adenyl)styrene oxide adducts at positions X6 in d(CGGACXAGAAG). d(CTTCTTGTCCG) and X7 in d(CGGACAXGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human n-ras protooncogene, were examined by 1H NMR. These were the S(61,2) and S(61,3) adducts. Chemical shift perturbations were in the 3'-direction from the sites of adduction; upfield changes associated with the styrene aromatic ring current were noted for S-SOA6 H2 and H1', T16 N3H, H6, and CH3 resonances in the S(61,2) oligomer. In the S(61,3) oligomer, S-SOA7 H1', T16 H1', C15 N4Ha, and H5 shifted upfield. The styrene aromatic rings flipped rapidly on the NMR time scale; under these conditions the ortho and meta aromatic protons were equivalent. A sequence effect, in which the S(61,2) adduct equilibrated between two conformers, while the S(61,3) adduct exhibited only a single conformation, was observed. Potential energy minimization of the S(61,2) adduct major conformation yielded a structure in which the styrene ring was oriented in the 3'-direction and interacted primarily with the complementary strand. For the S(61,3) adduct, 291 restraints were obtained from NOE data at three mixing times using relaxation matrix analysis. The emergent structures refined to an average rms difference of 1.3 A, determined by pairwise analysis. These were compared to NOE intensity data; the calculated sixth root residual index was 9.2 x 10-2 at 250 ms. In the refined structure, the styrene ring was also oriented in the 3'-direction and interacted with the complementary strand. The minor conformation of the S(61,2) adduct was not identified. These results contrasted with the diastereomeric R(61,2) and R(61,3) adducts, which underwent slow ring flips on the NMR time scale and for which small sequence effects involving the minimum energy conformation of the styrene ring were observed.

The pharmacologic evaluation of locomotor activity versus inflammatory parameters in rat adjuvant arthritis.

Adjuvant-induced arthritis (AA) is an experimental model of inflammatory joint disease in the rat which mimics rheumatoid arthritis. Although paw inflammation (e.g., swelling) is commonly used to monitor the efficacy of antiarthritic drugs, a reduction in locomotor function may provide a more sensitive evaluation of "functional disability" in AA rats. The purpose of the present study was to investigate the effect of dietary therapy with prednisolone or ibuprofen on locomotor activity as well as arthritic symptoms in established AA (days 20-42). AA rats demonstrated an increase in arthritis scores, spleen weights, fibrinogen, and WBC along with a reduction in locomotor function. Prednisolone (2 mg/kg/day) exhibited a positive therapeutic effect on all these parameters. Ibuprofen (50 mg/kg/day) consistently lowered arthritis scores and fibrinogen; however, locomotor function only improved on day 35. In conclusion, the measurement of locomotor activity in concert with other experimental parameters may provide a more meaningful evaluation of disease severity or improvement in AA.

An intracerebroventricular perfusion system developed for the study of centrally acting antihypertensive drugs in the rat.

A method for the complete construction and employment of an intracerebroventricular perfusion system for the rat brain is described. Rats maintained on the system for up to 7 hr displayed no detectable change in cardiovascular status. Penetration of intracerebroventricular-administered methylene blue dye into all areas of the cerebroventricles demonstrated the accessibility of the injection volume throughout the ventricular system. The hypotensive and bradycardic response produced by a 1.5-microgram/kg dose of clonidine (a centrally active antihypertensive agent) administered via the intracerebroventricular implant was of considerably greater magnitude and duration than an equivalent intravenous dose. Administration of [3H]-labeled clonidine via the intracerebroventricular implant resulted in relatively minor accumulation of the drug into peripheral organs, when compared to that found in the brain. These supportive data suggest the general applicability of this approach for studying the selective central effects of pharmacologic agents in a relatively inexpensive and easily procured laboratory animal.