Microneural anastomosis using cyanoacrylate adhesives.

We have developed a reliable method of microneural anastomosis using cyanoacrylate adhesives. This method involves overlapping the epineuriums of the two nerve ends and then applying two or three microdrops of cyanoacrylate adhesive on the surface of the epineurium just where the epineuriums overlap. The sciatic nerves of Sprague-Dawley rats were transected and repaired either with 10-0 nylon sutures or by using the described method. Histological evaluation showed no significant difference in the outcome of nerve regeneration between the two groups. It was concluded that the cyanoacrylate repair deserves to be considered as an alternative to the conventional suture technique in microneural anastomosis.

Cell-type specific signal transduction and gene regulation via mitogen-activated protein kinase pathway in catecholaminergic neurons by restraint stress.

It has been demonstrated that tyrosine hydroxylase (TH) gene is easily regulated in the CNS as well as peripheral nervous systems by stressful conditions. The stimuli, such as stress or reserpine administration, significantly increased the TH gene in noradrenergic neurons in the locus ceruleus (LC), but not in dopaminergic neurons in the substantia nigra (SN). To explore the molecular mechanisms governing differential TH gene regulation in catecholaminergic cells, the present study investigated the regulation of immediate early gene (c-Fos), transcription factors (pCREB, CREB binding protein [CBP]), mitogen-activated protein (MAP) kinases (phospho-extra-cellular regulated kinase [pERK]1/2, phospho-p38 MAP kinase [p-p38 MAPK], phospho-c-Jun N-terminal kinase [pJNK]) in the LC and SN in control conditions and in response to 2 h restraint stress (RS). Significant induction of c-Fos expression was observed in the LC, but not in the SN. In addition, pERK1/2 significantly increased following 2 h RS specifically in the LC, but not in the SN. No significant change was observed in p-p38 MAPK and pJNK. The expression of c-Fos and pERK1/2 preceded the upregulation of TH in the LC. Furthermore, pCREB and CBP also increased in the LC in response to 2 h RS. The induction of c-Fos prior to TH, in conjunction with the upregulation of pCREB and CBP in the LC, suggests that activator protein 1 and CRE transcription sites in the TH gene may be involved in the cell-type specific activation in the stress response, at least, by pERK1/2.

Dimethyldioxirane epoxidation of polycyclic fluoranthene hydrocarbons.

Direct epoxidation of polycyclic fluoranthenes with dimethyldioxirane provides several new mono- and di-oxide derivatives.

Probing the conformational heterogeneity of the acetylaminofluorene-modified 2'-deoxyguanosine and DNA by 19F NMR spectroscopy.

19F NMR spectroscopy was used to probe the conformation of a DNA adduct derived from the carcinogen 7-fluoro-N-acetyl-2-aminofluorene (FAAF) in three structural contexts: as a monomer and incorporated into single- and double-stranded DNA. The 19F NMR spectrum of dG-C8-FAAF [N-(deoxyguanosin-8-yl)-N-acetyl-7-fluoro-2-aminofluorene] in methanol at -30 degrees C exhibited four interconvertible signals in a 11:52:26:11 ratio. Dynamic NMR analysis indicated that the four torsional isomers arise from restricted rotation about the amide (gamma) (14.4 kcal/mol) and the guanyl-nitrogen (alpha) bonds. The conformational heterogeneity persisted in a single strand FAAF-12-mer, d(CTTCTTG[FAAF]ACCTC), whose 19F NMR spectrum at 22 degrees C and pH 7.0 gave only two signals in a 40:60 ratio, instead of four. The two 19F signals followed a two-site exchange with the rotation barrier of 14.7 kcal/mol about the amide (gamma') bond. A similar conformational theme was observed in the FAAF-12-mer duplex, d(CTTCTTG[FAAF]ACCTC).d(GAGGTCAAGAAG), which revealed two 19F resonances in a 41:59 ratio at 22 degrees C and pH 7.0. According to solvent-induced isotope and magnetic anisotropy effects, the two duplex conformers adopt exclusively a base displacement structure, being different only in their relative acetyl group orientations, cis (gamma' approximately 180 degrees) or trans (gamma' approximately 0 degrees ). Dynamic NMR data indicated that the two conformers do not exchange over a wide range of temperatures. This contrasts with the nonacetylated counterpart, which exhibits an equilibrium between the "B-type" and "stacked" conformers [Zhou, L., et al. (1997) J. Am. Chem. Soc. 119, 5384-5389]. The exclusive stacked nature of the AAF adducts may provide insight into why AAF adducts are more mutagenic and prone to repair than the nonacetylated AF adducts.

Nitroreduction of 4-nitropyrene is primarily responsible for DNA adduct formation in the mammary gland of female CD rats.

We determined whether DNA adducts derived from 4-nitropyrene (4-NP) are formed via nitroreduction or ring oxidation. DNA adduct markers derived from both pathways were prepared and, consequently, were compared with those obtained in vivo in rats treated with 4-NP. Following in vitro reaction of 9,10-epoxy-9,10-dihydro-4-nitropyrene (4-NP-9,10-epoxide), an intermediate metabolite derived from ring oxidation of 4-NP, with calf thymus DNA (average level of binding in two determinations was 8.5 nmol/mg of DNA), DNA was enzymatically hydrolyzed to deoxyribonucleosides and the DNA hydrolysates were analyzed by HPLC. Electrospray mass and 1H NMR spectra of the major products indicated that these adducts are deoxyguanosine (dG) derivatives that resulted from N2-dG substitution at the 9- or 10-position of the pyrene nucleus. However, these adducts were not detected in vivo in the rat mammary gland and liver following the administration of 4-NP. Nitroreduction of 4-NP catalyzed by xanthine oxidase in the presence of DNA resulted in three major putative DNA adducts (level of binding of 12.0 +/- 1.1 nmol/mg of DNA, n = 4) designated as peak 1 (46%), peak 2 (25%), and peak 3 (17%). Although peak 1 was further resolved into peaks 1a and 1b, both were unstable and gradually decomposed to peak 2, and the latter was unequivocally identified as pyrene-4,5-dione. On the basis of electrospray mass spectral analysis, peak 3 was tentatively identified as a deoxyinosine-derived 4-aminopyrene adduct. None of the adducts derived from nitroreduction of 4-NP catalyzed by xanthine oxidase coeluted with the synthetic standard N-(deoxyguanosin-8-yl)-4-aminopyrene prepared by reacting dG with N-acetoxy-4-aminopyrene. Nevertheless, HPLC analysis of the hydrolysates of liver and mammary DNA obtained from rats treated with [3H]-4-NP yielded four radioactive peaks, all of which coeluted with the markers derived from the nitroreduction pathway. These results indicate that nitroreduction is primarily responsible for DNA adduct formation in the liver and, especially, in the mammary gland which is the organ susceptible to carcinogenesis by this environmental agent.

Synthesis, characterization, and comparative conformational analysis of N-(deoxyguanosin-8-yl)aminopyrene adducts derived from the isomeric carcinogens 1-, 2-, and 4-nitropyrene.

Nitrated polycyclic aromatic hydrocarbons are mutagens/carcinogens that undergo in vivo activation by ring-oxidation and nitro-reduction pathways. We report the syntheses and comparative conformational analyses of N-(deoxyguanosin-8-yl)-n-aminopyrene adducts (dG-C8-n-AP, n = 1, 2, 4) derived from the three isomeric mononitropyrenes (1-, 2-, and 4-NP). The C8-amine nitrogens of these adducts have been enriched with 15N to examine the conformation about the pyrenyl-nitrogen and guanyl-nitrogen bonds that link the guanine and the pyrene moiety. These adducts are structurally isomeric, thus providing an interesting opportunity for systematic probing of the isomeric adduct conformations. Spectroscopic data indicated that the three isomeric aminopyrene adducts favor anti-glycosyl conformations, with C2'-endo (S) sugar puckering and a nearly planar conformation at the central amine nitrogen. The data further indicated differences in the extent of pi-electron conjugations about the pyrenyl-nitrogen bond, depending on the location of aminopyrene substitution. Thus while the 1- and 4-isomers both have substitution adjacent to a fused aromatic ring, the 2-isomer is highly symmetric and less sterically hindered. The 2-isomer adopts the most planar conformation, thereby having the most efficient pi-electron delocalization between the guanine and pyrene rings. The isomeric dG-C8-AP adducts and their nitro and amino precursors display physicochemical properties (HPLC retention time, UV pattern, 1H NMR data, mass fragmentation, etc.) distinctly dependent on their structures (1- and 4-isomers versus 2-isomer).

NMR structural studies of a 15-mer DNA duplex from a ras protooncogene modified with the carcinogen 2-aminofluorene: conformational heterogeneity.

Proton NMR studies were conducted on the complementary 15-mer DNA duplex, d(5'-TACTCTTCTT[AF]GACCT).d (5'-AGGTCAAGAAGAGTA) (designated as the AF-modified duplex). The sequence represents a portion of the mouse c-Ha-ras protooncogene and was selectively modified to contain a single N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) adduct at the deoxyguanosine corresponding to the first base of codon 61. The AF-modified duplex was found to exist in multiple conformations, with one being predominant (approximately 60%). The exchangeable and nonexchangeable protons belonging to the major conformer were sufficiently well-resolved to allow the assignment of the majority of the base and sugar protons. The one-dimensional proton spectra, as well as the NOE cross-peak patterns associated with this conformer of the AF-modified duplex both in H2O and D2O spectra, were strikingly similar to those observed for the major conformer of an analogous duplex containing N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) in the same position [Cho, B.P., Beland, F. A., & Marques, M. M. (1992) Biochemistry 31, 9587-9602]. The experimental results suggest that the AF- and ABP-modified duplexes adopt essentially identical major conformations, with each arylamine moiety being positioned in the major groove of a slightly disturbed B-type DNA duplex. Nonetheless, the absence of specific NOE cross peaks in the vicinity of the modification site indicates that the local structural perturbation is more severe in the AF-modified duplex. Although insufficient data precluded a detailed characterization of the minor conformers of the AF-modified duplex, the observation of significant shielding of the AF aromatic protons suggests a more dramatic structural alteration at the adduct site, possibly involving extensive stacking with the neighboring bases. The higher content (30-40%) of the minor conformers observed for the AF-modified duplex contrasted with the low contribution (5-10%) of similar structures in the ABP-modified duplex and may be attributed to a better overlapping efficiency of the planar AF ring with the nearby bases. Since the significant local perturbation observed in the minor conformers could provide a possible mechanism for mutations, our results support the view that the structural differences in the arylamine fragments of otherwise identical adducts have a direct influence on the conformational heterogeneities, which in turn may play a significant role in arylamine carcinogenesis.

NMR structural studies of a 15-mer DNA sequence from a ras protooncogene, modified at the first base of codon 61 with the carcinogen 4-aminobiphenyl.

Proton NMR studies were conducted on the complementary 15-mer duplex d(5'-TACTCTTCTTGACCT).(5'-AGGTCAAGAAGAGTA) (designated as unmodified 15-mer duplex) spanning a portion of the mouse c-Ha-ras protooncogene centered around codon 61. Identical studies were carried out on the same sequence, after specific modification with a reactive derivative of the carcinogen 4-aminobiphenyl (ABP), which resulted in incorporation of a single N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct in the noncoding strand (designated as ABP-modified 15-mer duplex). The adduct was located at the position corresponding to the first base of codon 61. The NMR data for the unmodified 15-mer duplex were fully consistent with a standard right-handed B-type DNA duplex conformation, with the possible exception of the frayed terminal base pairs. The ABP-modified 15-mer duplex was found to adopt one major conformation, although at least one additional conformation could be detected especially near room temperature. The major form, which exhibited strikingly similar NOE patterns as to those of the parent oligomer, both in H2O and D2O spectra, assumed a standard Watson-Crick base pairing throughout the entire length of the duplex, including the modification site and its flanking base pairs. Although some local perturbation of the helix could be detected in the vicinity of the modified guanosine, the NOE distance constraints established that the helix was globally right-handed and that the glycosidic torsion angles had the normal anti orientation, both at the modified base and its partner cytidine. Furthermore, the absence of strong NOE interactions between protons in the ABP moiety, which was rapidly rotating, and the nucleic acid protons was consistent with positioning of the arylamine moiety in the major groove of a weakly distorted double-helical structure. Although insufficient data prevented a detailed characterization of the minor conformer(s), the observation of significant shieldings for all the arylamine protons indicated a different orientation at the modified site in the minor contributor(s), possibly with extensive stacking between the ABP fragment and the neighboring bases.

Structure of oxidatively damaged nucleic acid adducts. 3. Tautomerism, ionization and protonation of 8-hydroxyadenosine studied by 15N NMR spectroscopy.

Natural abundance 15N NMR spectroscopy and ancillary spectroscopic techniques have been employed to study the solution structure of 8-hydroxyadenosine. 8-Hydroxyadenosine is a naturally occurring oxidized nucleic acid adduct that is generally implied to have an 8-hydroxy tautomeric structure. 15N NMR chemical shifts and coupling constants, however, indicate that the modified base exists as an 8-keto tautomer. The pH dependence of 15N NMR and UV spectra showed the presence of two pKa's, at 2.9 and 8.7, corresponding to protonation at N1 and ionization at N7, respectively. The latter results in the formation of an 8-enolate structure. Unusual upfield shifts of the 1H and 15N resonances of the NH2 group, and a reduction in the one-bond coupling constant 1JN6-H6, is indicative of an unfavorable steric or electronic interaction between the NH2 group and the adjacent N7-H proton. This interaction results in a subtle change in the structure of the NH2 group. In addition to being a possible mechanism for alteration of hydrogen bonding in oxidized DNA, this type of interaction gives a better understanding into N7-N9 tautomerism of adenine. Furthermore, the structure of 8-hydroxyadenosine has been related to possible mechanisms for mutations.