PubMed Health⌕ Search

Biomedical subjects

P F Swann

Publications and source records attributed to P F Swann.

At least 19 recordsLinked to original sources
PubMed2001

Isolation and sequencing of the cDNA of a novel cytochrome P450 from rat oesophagus.

RT-PCR was used to find whether cytochromes P450 of the 2A, 2B and 2E sub-families are expressed in the rat oesophagus. This showed that this tissue expresses a previously unknown member of the CYP2B sub-family, now designated CYP2B21. Using a combination of 5'- and 3'-RACE (rapid amplification of cDNA ends) and library screening, the cDNA was amplified and sequenced. The cDNA sequence (GenBank accession no. AF159245) covers the whole of the coding region and the whole of the 3'-untranslated region (UTR), but only 17 nt of the 5'-UTR. The DNA sequence has strong similarity to those of CYP2B1 and CYP2B2, with the derived amino acid sequence being 84 and 83% identical, respectively. The ease with which this cDNA was found in the cDNA library suggests that CYP2B21 is a major P450 of the oesophagus. The catalytic activity of this new CYP2B is not yet known, but as previous authors have reported that other members of this sub-family (CYP2B1 or 2B2) metabolize the selective oesophageal carcinogen N:-nitrosomethylbutylamine with the chemical selectivity necessary for carcinogenesis, i.e. they preferentially hydroxylate the alpha-carbon of the butyl chain, this new CYP2B may be the nitrosamine-activating enzyme of the oesophagus.

Amino Acid Sequence↗
PubMed2000

Thymine-DNA glycosylase and G to A transition mutations at CpG sites.

About 23% of mutations in hereditary human diseases and 24% of mutations in p53 in human cancers are G to A transitions at sites of cytosine methylation suggesting that these sites are either foci for DNA damage, or foci for damage that is poorly repaired. Thymine produced at these sites by the hydrolytic deamination of 5-methylcytosine is removed by thymine-DNA glycosylase. Thymine-DNA glycosylase will also remove 3,N(4)-ethenocytosine and uracil from DNA. The action of this enzyme is limited by its very low k(cat) and by tight binding to the apurinic site produced when the thymine is removed. These properties of the enzyme suggest that the inefficiency of the base excision repair pathway that it initiates may be the underlying cause of the prevalence of these mutations.

DNA Damage↗
PubMed1999

Human thymine DNA glycosylase binds to apurinic sites in DNA but is displaced by human apurinic endonuclease 1.

In vitro, following the removal of thymine from a G.T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (koff 1.8 - 3.6 x 10(-5) s-1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C.AP and SMeG.AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C.U and SMeG.T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G.AP sites sufficiently to allow it to remove more than one thymine from G.T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase beta, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.

Base Pair Mismatch↗
PubMed1998

Kinetics of the action of thymine DNA glycosylase.

The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: kcat values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0. 0046, and 0.0013 min-1, respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S6-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: kcat values for removal of thymine from SMeG.T with C.G, T. A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0. 0010 min-1, respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i.e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with SMeG.T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG.T with TpSMeG.T), and so these contacts are probably cooperative. The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.

Base Composition↗
PubMed1997

Cytotoxic mechanism of 6-thioguanine: hMutSalpha, the human mismatch binding heterodimer, binds to DNA containing S6-methylthioguanine.

It has been suggested that the cytotoxicity of 6-thioguanine depends upon (1) incorporation of 6-thioguanine into DNA, (2) methylation by S-adenosylmethionine (SAM) of the thio group to give S6-methylthioguanine, (3) miscoding during DNA replication to give [SMeG] x T base pairs, and (4) recognition of these base pairs by proteins of the postreplicative mismatch repair system. Here we have investigated systematically the ability of proteins present in human cell extracts to bind to DNA containing S6-methylthioguanine. We found that [SMeG] x T base mismatches were bound by the mismatch binding complex, hMutS alpha, and that the level of binding was dependent upon the base 5' to the S6-methylthioguanine in the order G > C = A > T. Extracts from cells that lack either hMSH2 (LoVo cells) or GTBP (HCT-15 cells), two components of the hMutS alpha complex, were unable to bind the [SMeG] x T base pair. We also found that hMutS alpha was able to bind to [SMeG] x C base pairs when the S6-methylthioguanine was in the sequence 5'-Cp[SMeG]. This suggests that miscoding by S6-methylthioguanine residues in DNA during DNA synthesis may not be an absolutely required step in the mechanism of cytotoxicity. Also, since CpG sequences are so important in gene regulation, this result may be of considerable significance.

Base Composition↗
PubMed1997

Opium and oesophageal cancer: effect of morphine and opium on the metabolism of N-nitrosodimethylamine and N-nitrosodiethylamine in the rat.

The high incidence of oesophageal cancer in Northern Iran has been associated with opium. N-Nitrosamines are the only carcinogens known to induce oesophageal cancer in animals. Ethanol, which is the major influence on oesophageal cancer incidence in the West, inhibits the first pass clearance of N-nitrosodimethylamine in animals and increases the alkylation of oesophageal DNA by oesophageal cancer-inducing N-nitrosamines. The experiments now reported were to test whether opium or morphine, which is the major alkaloid in opium, have a similar effect. It is shown that administration of morphine to rats does increase the ethylation of oesophageal DNA by N-nitrosodiethylamine and may reduce the first pass clearance of N-nitrosodimethylamine by the liver, but only at high doses of morphine.

Administration, Oral↗
PubMed1996

Role of postreplicative DNA mismatch repair in the cytotoxic action of thioguanine.

It is proposed here that the delayed cytotoxicity of thioguanine involves the postreplicative DNA mismatch repair system. After incorporation into DNA, the thioguanine is chemically methylated by S-adenosylmethionine to form S6-methylthioguanine. During DNA replication, the S6-methylthioguanine directs incorporation of either thymine or cytosine into the growing DNA strand, and the resultant S6-methylthioguanine-thymine pairs are recognized by the postreplicative mismatch repair system. Azathioprine, an immunosuppressant used in organ transplantation, is partly converted to thioguanine. Because the carcinogenicity of N-nitrosamines depends on formation of O6-alkylguanine in DNA, the formation of the analog S6-methylthioguanine during azathioprine treatment may partly explain the high incidence of cancer after transplantation.

Animals↗
PubMed1994

Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond.

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand. Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1993

Clearance of N-nitrosodimethylamine and N-nitrosodiethylamine by the perfused rat liver. Relationship to the Km and Vmax for nitrosamine metabolism.

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Chromatographic separation of oligodeoxynucleotides with identical length: application to purification of oligomers containing a modified base.

A general procedure is described for separation and purification of oligodeoxynucleotides of identical length but different base composition, in particular, of oligomers containing modified bases such as 4-substituted thymines and 6-substituted guanines, using an anion-exchange column (either Mono Q or NucleoPac). The modified oligomers can be well separated from the analogous oligomers containing unmodified thymine or guanine under the basic conditions of the chromatography. The effects of oligomer length, base composition, and lipophilicity on the separation are discussed. A general rule which can be used for prediction of the order of elution of different oligomers and for estimation of tautomeric form of a modified base in the oligomer is presented.

Anion Exchange Resins↗
PubMed1992

Molecular dynamics of alkylated DNA.

The effect of methylation of the O4 atom of thymine in two oligonucleotide sequences is investigated by molecular dynamics simulations. Three types of environments are considered including: (i) in vacuo calculation, with a distance-dependent dielectric function and unhydrated counter-ions; (ii) in vacuo calculation, with a distance-dependent dielectric constant and hydrated counter-ions; and (iii) with a 9 A thick explicit water layer and counter-ions. In all environments, the oligonucleotide sequence containing the chemically modified thymine paired with guanine is more stable than the oligonucleotide sequence in which the modified thymine is paired with adenine. The methyl group attached to the O4 atom of thymine is found in a syn configuration with respect to the N3 atom. The best fit between the experimental NMR results and the molecular dynamics simulations is obtained using the environment with hydrated counter-ions.

Alkylation↗
PubMed1991

Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility.

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 61st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing O4-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1991

Nitrosamine-induced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras.

Mammary and skin tumors induced in rodents by N-methyl-N-nitrosourea treatment have a G:C to A:T transition mutation in codon 12 of H-ras, probably resulting from alkylation of O6 of guanine by the carcinogen. This codon contains two guanines (5'-GGA-3'), but mutations are observed only in the central base pair of this codon. The same selectivity for mutations of -GGA-sequences has also been observed in Escherichia coli. It is known that the central G in the sequence GGA is a preferred site for alkylation, but the magnitude of chemical selectivity is insufficient to provide a complete explanation for the biological observation which is still unexplained. We have measured accurate rates of repair by the E. coli and gene O6-alkylguanine-DNA-alkyltransferase of an O6-methylguanine in various positions in chemically synthesized 15-base pair DNA duplexes having the H-ras sequence. The rate of repair varied 25-fold, depending on the sequence flanking the methylguanine. An O6-methylguanine in position 2 of codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation for the selective mutation of this position. Using an antibody to probe the accessibility of the O6-methyldeoxyguanosine, it was shown that the rate of repair is a reflection of the conformation of the sequence containing the alkylated base, because the avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the methylguanine, with the most rapidly repaired O6-methylguanines being those most easily bound by the antibody.

Animals↗
PubMed1990

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.

Alkylation↗
PubMed1990

Conformational transitions in thymidine bulge-containing deoxytridecanucleotide duplexes. Role of flanking sequence and temperature in modulating the equilibrium between looped out and stacked thymidine bulge states.

Structural features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimensional NMR analysis of through-bond and through-space connectivities in the otherwise self-complementary d(C-C-G-T-G-A-A-T-T-C-C-G-G) (GTG 13-mer) and d(C-C-G-G-A-A-T-T-C-T-C-G-G) (CTC 13-mer) duplexes in aqueous solution. These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 degrees C), whereas the equilibrium shifts in favor of the stacked state at elevated temperatures (35 degrees C) prior to the onset of the duplex-strand transition. By contrast, the extra thymidine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.

Base Sequence↗
PubMed1990

Crystal structure and sequence-dependent conformation of the A.G mispaired oligonucleotide d(CGCAAGCTGGCG).

The crystal structure of the dodecanucleotide d(CGCAAGCTGGCG) has been determined to a resolution of 2.5 A and refined to an R factor of 19.3% for 1710 reflections. The sequence crystallizes as a B-type double helix, with two G(anti).A(syn) base pairs. These are stabilized by three-center hydrogen bonds to pyrimidines that induce perturbations in base-pair geometry. The central AGCT region of the helix has a wide (greater than 6 A) minor groove.

Adenine↗
PubMed1990

Solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine.

Practical methods for the routine solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine are described. It is shown that if the 2-amino group of the alkylated base is protected with a phenylacetyl group, and a mixture of nitrobenzaldoximate ions and ammonia used to remove the protecting groups from the oligomer at the end of the synthesis, there is negligible formation of 2,6-diaminopurine and that after chromatography pure oligomers are obtained.

Base Sequence↗