Postsynthetic generation of a major acrolein adduct of 2'-deoxyguanosine in oligomeric DNA.
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Acrolein↗
Biomedical subjects
F Johnson
Publications and source records attributed to F Johnson.
Immunoaffinity/gas chromatography/high-resolution mass spectrometry method for the detection of N(2),3-ethenoguanine.
Etheno adducts are formed after exposure to a number of carcinogens, including vinyl chloride, as well as endogenously as a result of lipid peroxidation. A sensitive and selective assay for N(2), 3-ethenoguanine (epsilonGua) was developed using immunoaffinity (IA) columns made with polyclonal antibodies to epsilonGua followed by gas chromatography/electron capture negative chemical ionization/high-resolution mass spectrometry (GC/ECNCI/HRMS) analysis of its pentafluorobenzyl derivative. These IA columns were specific for epsilonGua and did not bind guanine, deoxyguanosine, 1, N(6)-ethenoadenine, or 1,N(2)-ethenoguanine. The level of recovery of standards from the IA columns was 107 +/- 7% and throughout the entire method (using nucleoside enzymatic digestion) with or without DNA was 72 +/- 6%. Four different hydrolysis/digestion procedures were compared, nucleoside enzymatic (EZ), neutral thermal hydrolysis (NT), formic acid hydrolysis (FA), and HCl hydrolysis. All hydrolysis methods with subsequent IA chromatography produced linear standard curves with r(2) values of 0.999 or better. The level of epsilonGua in chloroethylene oxide-treated calf thymus DNA (CEO-ctDNA) was 38 +/- 2, 42 +/- 3, and 49 +/- 2 fmol of epsilonGua/microg of DNA using EZ, NT, and FA, respectively. These numbers remained consistent when the amount of DNA processed was doubled or tripled. These numbers were comparable to the previously published value of 55 +/- 8 fmol of epsilonGua/micrograms of DNA for the same DNA using HCl hydrolysis, cation exchange cleanup, and LC/MS analysis [Yen, T. Y., et al. (1996) J. Mass Spectrom. 31, 1271-1276]. Additionally, HCl hydrolysis of rat liver DNA from control and vinyl fluoride-exposed rats gave similar epsilonGua results when compared to those from enzymatic digestion using this method. This method gave a detection limit of 5 epsilonGua adducts/10(8) normal dGuo nucleosides in 150 micrograms of DNA using EZ and somewhat lower detection limits using NT and HCl hydrolysis. The method is more sensitive and selective than previously used methods for the quantitation of this adduct.
Animals↗
8-Amino-2'-deoxyguanosine incorporation into oligomeric DNA.
During the incorporation of 8-amino-dG into oligomeric DNA, the deprotection conditions previously recommended (28% ammonia at room temperature) do not effect complete removal of the dimethylaminomethylene protecting groups. At elevated temperatures oxidative degradation of the oligomer and exchange of ammonia with dimethylamine in the protecting group at C8 occurred. The resolution of these problems and a method to obtain a series of homogeneous oligomers in reasonable yield containing 8-amino-dG located site-specifically are described.
Base Sequence↗
Intense pulsed light treatment of hirsutism: case reports of skin phototypes V and VI.
Removal of unwanted hair is a common cosmetic concern. For hirsute women, treatment often requires drug therapy and various methods to physically remove the hair. Traditional methods of hair removal include shaving, waxing, tweezing, depilatory creams and electrolysis. Hair removal methods based on light technology, such as lasers and intense pulsed light systems, are alternative methods for longer-term hair removal. Intense pulsed light has been used in our clinic during the past 2 years to treat light-to-dark skinned patients, including skin types V and VI. We present here the treatment, using an intense pulsed light source, of three dark skinned patients with hirsutism. Patients were treated during multiple sessions (five to seven) for unwanted facial hair. Sessions were conducted monthly and patients were evaluated at follow-up sessions 2-7 months after the final treatment. Successful clearance of unwanted hair was achieved in all three patients with no pigmentary changes observed during the final follow-up sessions. Folliculitis and hyperpigmentation from tweezing were also treated by the intense pulsed light source. These results suggest that intense pulsed light is an effective source for hair removal and may, with proper parameter selection, be useful in the treatment of very dark skin types.
Adult↗
Single-stranded oligodeoxyribonucleotides are substrates of Fpg protein from Escherichia coli.
The interaction of Escherichia coli Fpg protein, which catalyzes excision of several damaged purine bases including 8-oxoguanine (oxoG) from DNA with a set of single- (ss) and double-stranded (ds) 23-mer oligodeoxyribonucleotides (ODNs) containing 8-oxoguanine(s) at various positions, has been investigated. The affinities of different ss ODNs (KM = 0.55-1.3 microM) were shown to be 12-170 times less than those for corresponding ds ODNs (KM = 6-60 nM). Depending on the position of the oxoG within the ODNs, relative initial rates of conversion of ss substrates may be less than, comparable, or greater than those for ds ODNs. The enzyme can remove 5'-terminal oxoG from ODNs only if the 5'-end is phosphorylated. Fpg does not release oxoG residues from the ultimate and penultimate 3'-terminal positions. Duplexes containing two adjacent oxoG are poor substrates for the glycosylase.
DNA↗
Is anaesthesia evidence-based? A survey of anaesthetic practice.
We were interested in measuring the proportion of anaesthetic interventions in routine practice that are supported by evidence in the literature. We surveyed our hospital practice, asking anaesthetists to nominate a primary problem (if any) and their chosen intervention. Each intervention was classified into one of four levels according to the strength of the evidence recovered from the literature. We found that 96.7% were evidence-based (levels I-IV), including 32% supported by randomized, controlled trials (levels I and II). These results are similar to recent studies in other specialties and refute the claim that only 10-20% of treatments have any scientific foundation.
Anesthesia↗
Formation of s-triazines during aerial oxidation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in concentrated ammonia.
After automated DNA synthesis, oligodeoxynucleotides containing 8-oxoguanine are sensitive to aerial oxidation when subjected to the basic conditions necessary for deprotection and release of the oligomer from the control pore glass support. The major oxidation products of this heterocyclic moiety have been characterized by permitting 8-oxo-7,8-dihydro-2'-deoxyguanosine to react with oxygen in the presence of 28% aqueous ammonia at room temperature. Products were isolated by reverse phase HPLC and analyzed by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry of the trimethylsilyl-derivatives. 2-Amino-4-hydroxy-s-triazine-6-carboxylic acid and 2-amino-4-hydroxy-6-carbamyl-s-triazine were identified by these techniques and standards were synthesized. In addition, GC-MS analysis revealed other oxidation products, including urea, guanidine and 2-deoxyribose, which were not observed by HPLC because these compounds are transparent in the UV region of the spectrum. Both s-triazines were also observed when a purified, synthetic oligodeoxynucleotide containing a single 8-oxoguanine moiety was exposed to the same conditions. Oxidation of 8-oxoguanine appears to parallel the uric acid oxidation pathway, and a mechanistic scheme is proposed to account for the products of degradation.
8-Hydroxy-2'-Deoxyguanosine↗
Endoscopic assessment of oesophagitis: clinical and functional correlates and further validation of the Los Angeles classification.
BACKGROUND: Endoscopic oesophageal changes are diagnostically helpful and identify patients exposed to the risk of disease chronicity. However, there is a serious lack of agreement about how to describe and classify the appearance of reflux oesophagitis AIMS: To examine the reliability of criteria that describe the circumferential extent of mucosal breaks and to evaluate the functional and clinical correlates of patients with reflux disease whose oesophagitis was graded according to the Los Angeles system. METHODS: Forty six endoscopists from different countries used a detailed worksheet to evaluate endoscopic video recordings from 22 patients with the full range of severity of reflux oesophagitis. In separate studies, Los Angeles system gradings were correlated with 24 hour oesophageal pH monitoring (178 patients), and with clinical trials of omeprazole treatment (277 patients). RESULTS: Evaluation of circumferential extent of oesophagitis by the criterion of whether mucosal breaks extended between the tops of mucosal folds, gave acceptable agreement (mean kappa value 0.4) among observers. This approach is used in the Los Angeles system. An alternative approach of grouping the circumferential extent of mucosal breaks as occupying 0-25%, 26-50%, 51-75%, 76-99%, or 100% of the oesophageal circumference, gave unacceptably high interobserver variation (mean kappa values 0-0.15) for all but the lowest category of extent (mean kappa value 0.4). Severity of oesophageal acid exposure was significantly (p<0.001) related to the severity grade of oesophagitis. Preteatment oesophagitis grades A-C were related to heartburn severity (p<0.01), outcomes of omeprazole (10 mg daily) treatment (p<0.01), and the risk for symptom relapse off therapy over six months (p<0.05). CONCLUSIONS: Results add further support to previous studies for the clinical utility of the Los Angeles system for endoscopic grading of oesophagitis.
Endoscopy, Digestive System↗
Validation of a quantitative method for defining CAD/CAM socket modifications.
A quantitative method was developed for defining manual socket modifications, averaging these modifications over a series of amputees, and using the average modifications as a template in commercial CAD/CAM systems. The CADVIEW programme (i.e. software for viewing and analysing CAD sockets) was rewritten to provide comparison functions for aligning sockets to a common axis, visualising the differences between sockets, generating modification outlines, assigning apex point values, and averaging the modification outlines. A CAD template generated in this manner should be the best general representation of a prosthetist's modification style. To test this hypothesis, 13 people with trans-tibial amputations were fitted with both a manual and a CAD/CAM socket. Questionnaires were completed by the subjects and by the prosthetist to obtain information on prosthetic comfort, function, and overall satisfaction. Ground reaction force and stride parameter data were also collected for each prosthesis during gait laboratory testing. No significant differences were found between the manually designed socket and the CAD/CAM designed socket for all data except the vertical peak forces on the amputated side. These results support the clinical application of this quantitative technique for making the transition from manual to CAD/CAM prosthetic modification procedures.
Adult↗
Cellular response to exocyclic DNA adducts.
The mutagenic potential of three exocyclic DNA adducts was studied in Escherichia coli and simian kidney cells by incorporating them into single-stranded DNA. Differences in the mutagenic potency of the adducts were observed between hosts: 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine were more mutagenic in simian cells, whereas 1,N2-(1,3-propan-1,3-diyl)-2'-deoxyguanosine was more mutagenic in E. coli. To investigate the cellular response to DNA adducts, a double-stranded DNA vector system was developed. Use of this system showed that 1,N6-ethenodeoxyadenosine blocks DNA synthesis strongly, and DNA synthesis past this adduct was highly accurate in E. coli. The blockage of DNA synthesis was overcome in an error-free manner by the recombination repair mechanism (daughter-strand gap repair).
Animals↗
The impact of an exocyclic cytosine adduct on DNA duplex properties: significant thermodynamic consequences despite modest lesion-induced structural alterations.
The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.
Base Sequence↗
Suppression of adventitious formation of 8-oxoguanine(TMS)4 from guanine during trimethylsilylation.
GC/MS quantitation of 8-oxoguanine derived from DNA may yield artificially high values when trimethylsilylation is used as the technique to form a volatile derivative. Significant quantities of the tetrakis trimethylsilyl-derivative of 8-oxoguanine may be formed from guanine during the derivatization reaction at elevated temperatures. We have screened eight antioxidants in an attempt to identify a substance that will reduce the spurious formation of this product. Whereas several compounds were active, N-phenyl-1-naphthylamine was the most effective agent. Moreover, this compound was sufficiently basic to act as a catalyst for the derivatization reaction. Our data, which are based on the use of an isotopically labeled internal standard, show that the level of the tetrakis trimethylsilyl derivative of 8-oxoguanine remained constant as a function of derivatization heating time when N-phenyl-1-naphthylamine was added to a reaction in which guanine was present. In contrast, the level in control samples increased linearly with reaction time indicating formation of 8-oxoGua during derivatization. The level of 8-oxoguanine was measured in both a synthetic oligodeoxynucleotide containing a single 8-oxo-2'-deoxyguanosine moiety and commercially available calf thymus DNA using this technique.
1-Naphthylamine↗
Thermodynamic consequences of an abasic lesion in duplex DNA are strongly dependent on base sequence.
The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C.G or G.C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magnitude of this impact being strongly dependent on sequence context.
Base Composition↗
Benefits of colorectal cancer patient follow-up.
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Colorectal Neoplasms↗
Synthesis of enzymatically noncleavable carbocyclic nucleosides for DNA-N-glycosylase studies.
Carbocyclic nucleosides have been of great interest as antiviral agents and in studies in the area of antisense technology. The recent finding that the replacement of a single 2'-deoxynucleoside in DNA by a carba analogue does not alter the Watson-Crick base pairing, yet at the same time provides a chemically and enzymatically stable "glycosidic" linkage, led us to examine this class of compound as enzyme inhibitors of the DNA-repair enzymes involved in oxidative damage. We now report the synthesis and incorporation into oligomeric DNA via suitable derivatives, the carbanucleosides 8-oxo-7,8-dihydro-2'-deoxycarbainosine, 8-oxo-7,8-dihydro-2'-deoxycarbaguanosine, and 2'-deoxyaristeromycin. Aristeromycin (1) was deoxygenated at the 2'-position as follows. Treatment of 1 with TPDSCl2 gave the 3',5'-protected derivative 3 (76%) which on phenylthiocarbonylation at the 2'-position gave 4 in 51% yield. The latter compound on reduction with Bu3SnH led to the 2'-deoxy derivative 5 (90%). Benzoylation followed by deprotection with TBAF in THF then gave the desired intermediate (6) in 65% yield. N2-Isobutyryl-8-oxo-7,8-dihydro-2'-deoxycarbaguanosine (16) was synthesized from 3-chloro-2'-deoxycarbainosine (9). Treatment of 9, either with hydrazine followed by catalytic reduction of the 2-hydrazino derivative or with 1-(2-nitrophenyl)ethylamine followed by photolysis of the resulting 2-substituted derivative, in both instances gave the desired 2'-deoxycarbaguanosine (12) in approximately 50% overall yield in each case. Bromination of 12 gave 13 (90%) which, when treated with BnONa in DMSO at 65 degrees C, led to the 8-benzyloxy derivative 14 (46%). Isobutyrylation of 14 followed by catalytic reduction then afforded 16. 8-Oxo-7,8-dihydro-2'-deoxycarbainosine (23) was prepared in four steps. Bromination of 2'-deoxyaristeromycin (19) at the 8-position gave 20 (> 95%) which was converted to the 8-benzyloxy derivative 21 (61%) using BnONa/DMSO at 80 degrees C. Reductive debenzylation of 21 then led to 8-oxo-7,8-dihydro-2'-deoxyaristeromycin (approximately 100%) which, when treated with adenosine deaminase, provided the desired carbainosine derivative 23 in quantitative yield. Compounds 6, 16, and 23 were converted to their respective 5'-O-DMT, 3'-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine] derivatives (8, 18, and 25) in excellent overall yields. The latter were then used to synthesize a series of DNA oligomers by automated procedures.
Adenosine↗
Members of the olfactory receptor gene family are contained in large blocks of DNA duplicated polymorphically near the ends of human chromosomes.
We have identified three new members of the olfactory receptor (OR) gene family within a large segment of DNA that is duplicated with high similarity near many human telomeres. This segment is present at 3q, 15q, and 19p in each of 45 unrelated humans sampled from various populations. Additional copies are present polymorphically at 11 other subtelomeric locations. The frequency with which the block is present at some locations varies among populations. While humans carry seven to 11 copies of the OR-containing block, it is located in chimpanzee and gorilla predominantly at a single site, which is not orthologous to any of the locations in the human genome. The observation that sequences flanking the OR-containing segment are duplicated on larger and different sets of chromosomes than the OR block itself demonstrates that the segment is part of a much larger, complex patchwork of subtelomeric duplications. The population analyses and structural results suggest the types of processes that have shaped these regions during evolution. From its sequence, one of the OR genes in this duplicated block appears to be potentially functional. Our findings raise the possibility that functional diversity in the OR family is generated in part through duplications and inter-chromosomal rearrangements of the DNA near human telomeres.
Amino Acid Sequence↗
The preterm prediction study: the value of new vs standard risk factors in predicting early and all spontaneous preterm births. NICHD MFMU Network.
OBJECTIVES: This study was undertaken to determine the relationship between fetal fibronectin, short cervix, bacterial vaginosis, other traditional risk factors, and spontaneous preterm birth. METHODS: From 1992 through 1994, 2929 women were screened at the gestational age 22 to 24 weeks. RESULTS: The odds ratios for spontaneous preterm birth were highest for fetal fibronectin, followed by a short cervix and history of preterm birth. These factors, as well as bacterial vaginosis, were more strongly associated with early than with late spontaneous preterm birth. Bacterial vaginosis was more common--and a stronger predictor of spontaneous preterm birth--in Black women, while body mass index less than 19.8 was a stronger predictor in non-Black women. This analysis suggests a pathway leading from Black race through bacterial vaginosis and fetal fibronectin to spontaneous preterm birth. Prior preterm birth is associated with spontaneous preterm birth through a short cervix. CONCLUSIONS: Fetal fibronectin and a short cervix are stronger predictors of spontaneous preterm birth than traditional risk factors. Bacterial vaginosis was found more often in Black than in non-Black women and accounted for 40% of the attributable risk for spontaneous preterm birth at less than 32 weeks.
Cervix Uteri↗
Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine.
Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.
DNA↗