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J H van Boom

Publications and source records attributed to J H van Boom.

At least 73 records · Page 4Linked to original sources
PubMed1994

The solution structure of the circular trinucleotide cr(GpGpGp) determined by NMR and molecular mechanics calculation.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.

Guanine↗
PubMed1994

Assignment strategies and analysis of cross-peak patterns and intensities in the three-dimensional homonuclear TOCSY-NOESY of RNA.

The application of 3D TOCSY-NOESY to the analysis of RNA is presented, using a TOCSY-NOESY spectrum of the RNA duplex r(5'GGGCUGAAGCCU'). It is shown that for RNA molecules, 3D spectra can be obtained with a digital resolution comparable to that obtained for 2D NMR with full spectral information. The improvement in assignment over 2D methods is shown and discussed on the basis of an assignment strategy presented earlier. A simple and straightforward method for determining sugar puckers and gamma backbone torsion angles is presented, which is derived from an analysis of cross-peak intensities originating from the TOCSY coherence transfer among sugar protons and H5'/5" protons. The stereospecific assignment of the H5'/5" resonances in 3D TOCSY-NOESY spectra is also discussed.

Image Processing, Computer-Assisted↗
PubMed1993

Structure of the complex of lac repressor headpiece and an 11 base-pair half-operator determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics.

The structure of the complex of lac repressor headpiece and an 11 base-pair lac half-operator has been determined by NMR spectroscopy and restrained Molecular Dynamics calculations. In total 508 distances were derived from two-dimensional nuclear Overhauser enhancement measurements, 260 of which are within the headpiece, 212 within the operator and 36 between operator and headpiece. An equilibrium restrained Molecular Dynamics calculation of the complex in aqueous solution, spanning 85 picoseconds, has been used to analyze the structure. Configuration sampling by an annealing procedure has been undertaken as well in order to estimate the precision of the structure determination. Our data confirm the results of previous two-dimensional NMR studies that the orientation of the recognition helix of lac repressor in the major groove of DNA with respect to the operator dyad axis is opposite to the orientation found in complexes of other DNA binding proteins of the helix-turn-helix class. We find a number of tight contacts between the protein and the operator that are in agreement with the available genetic and biochemical data. The anchoring of lac headpiece on the operator is similar to that of other repressors. Other features are unique for lac headpiece: relative few direct hydrogen bonds between side-chains and bases; extensive apolar contacts; many direct and water-bridged contacts to phosphates from residues in or close to the recognition helix. Overall, an interconnected set of interactions is observed, involving base-specific contacts, phosphate contacts, intra-protein and water-bridged hydrogen bonds. Several of these interactions appear to be dynamic, i.e. fluctuating in time, rather than static.

Amino Acid Sequence↗
PubMed1993

Exploring the DNA binding domain of gene V protein encoded by bacteriophage M13 with the aid of spin-labeled oligonucleotides in combination with 1H-NMR.

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

Amino Acid Sequence↗
PubMed1993

Molecular structure of the A-tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor groove binding drug netropsin.

The molecular structure of the complex between the minor groove binding drug netropsin and the dodecamer d(CGCAAATTTGCG) has been solved and refined by X-ray diffraction analysis to an R-factor of 19.8% and 2.2-A resolution. The drug lies in the narrow minor groove of the B-DNA fragment, covering five of the six A.T base pairs (from A5.T20 to T9.A16). The long six A.T base pair tract allows the drug to bind in a position that optimizes its contacts with the DNA, establishing hydrogen bonds with O2 of thymines and N3 of adenines. The DNA molecule shows a high propeller twist only at the A6.T19 step of the A-tract. Two three-centered hydrogen bonds are observed in the major groove at half of the A-tract.

Base Sequence↗
PubMed1993

Structural effects of the C2-methylhypoxanthine:cytosine base pair in B-DNA: A combined NMR and X-ray diffraction study of d(CGC[m2I]AATTCGCG).

C2-Methylhypoxanthine (m2I) is a synthetic analog of guanine with the N2-amino group replaced by a methyl group. We have studied the structural consequence of the m2I incorporation in DNA by a combination of X-ray crystallographic, NMR, and enzymatic analyses. The crystal structure of d(CGC[m2I]AATTCGCG) has been solved and refined to an R factor of 20.7% at 2.25-A resolution. In the DNA duplex, the two independent m2I:C base pairs maintain the Watson-Crick scheme. While the C2-methyl group of m2I is in van der Waals contact with the O2 of the base-paired cytosine, it only causes the base pair to have slightly higher propeller twist and buckle angles. Its solution structure was analyzed by the NMR refinement procedure SPEDREF [Robinson, H., & Wang, A. H.-J. (1992) Biochemistry 31, 3524-3533] using 2D nuclear Overhauser effect data. Two starting models, a relaxed fiber model and an X-ray model, were subjected to the NOE-constrained refinement using 1518 NOE cross-peak integrals to arrive at the final models with (NOE) R factors of 13.8% and 14.3%, respectively. The RMSD between the two refined models (all atoms included) is 1.23 A, which presently seems to be near the limit of convergence of NOE-based refinement. The local structures of the two models are in better agreement as measured by the RMSD of the dinucleotide steps, falling in the range 0.54-0.98 A. Both refined solution structures confirm that the m2I dodecamer structure is of the B-DNA type with a narrow minor groove at the AT region, as observed in the crystal. However, significant differences exist between the crystal and solution structures in parameters such as pseudorotation angles, propeller twist angles, etc. The solution structure tends to have a more uniform backbone conformation, an observation consistent with that concluded from the laser Raman study of d(CGCAAATTTGCG) [Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., & Thomas, G. J., J. (1988) Biochemistry 27, 931-938]. Three related dodecamers, d(CGCGAATTCGCG), d(CGC[m2I]AATTCGCG), and d(CGC[e6G]AATTCGCG), were tested as substrates for the restriction endonuclease EcoRI. The m2I dodecamer was active, but the e6G dodecamer was not. Our results illustrate the complementarity in terms of the structural information provided by the two methods, X-ray diffraction and NMR.

Base Composition↗
PubMed1993

The application of the AMB protective group in the solid-phase synthesis of methylphosphonate DNA analogues.

Partially methylphosphonate-modified oligodeoxynucleotides were synthesized on solid-phase by employing the easily removable 2-(acetoxymethyl)benzoyl (AMB) group as base-protecting group. Although a rapid AMB deprotection can be accomplished in methanolic potassium carbonate, the lability of the methylphosphonate linkage towards potassium carbonate/methanol excludes the use of this deprotection reagent. Thus, saturated ammonia solution in methanol was investigated as an alternative reagent for AMB removal. It is demonstrated that the combination of the AMB protective group and ammonia/methanol as deprotection reagent significantly improves the synthesis of methylphosphonate-modified DNA fragments. A mild overnight treatment at room temperature is sufficient for complete removal of the AMB group, whereas deprotection of conventionally protected oligonucleotides requires much longer exposure to basic conditions at elevated temperatures.

Acetates↗
PubMed1993

Simultaneous incorporations of two anticancer drugs into DNA. The structures of formaldehyde-cross-linked adducts of daunorubicin-d(CG(araC)GCG) and doxorubicin-d(CA(araC)GTG) complexes at high resolution.

Anthracycline antibiotics (notably daunorubicin (DAU) and doxorubicin (DOX)) and nucleoside analog arabinosylcytosine (araC or aC) are important anticancer drugs. They are sometimes used together in the treatment of certain cancers. Both classes of compounds act by blocking DNA replication and transcription. To probe whether both drugs can be incorporated simultaneously into DNA and the possible structural consequences, we carried out x-ray diffraction analyses of the complexes between DAU/DOX and araC-containing DNA hexamers cross-linked with formaldehyde. The crystal structures were determined to high resolution (DAU-CGaCGCG, 1.2 A, space group P4(1)2(1)2, R = 0.182, 3275 reflections; DOX-CAaCGTG, 1.5 A, space group C2, R = 0.175, 3359 reflections), and they are similar to those of the previously studied DAU- and DOX-DNA complexes, despite different crystal packings. Two DAU/DOX molecules intercalate at both ends of the helix with their amino sugars in the minor groove. As in the structure of DAU-CGCGCG (Wang, A.H.-J., Gao, Y.-G., Liaw, Y.-C., and Li, Y.K. (1991) Biochemistry 30, 3812-3815), a covalent methylene bridge (from formaldehyde) between the N3' of daunosamine and the N2 of the guanine is formed in both adducts. In DOX-CAaCGTG, the two halves are slightly different with a root-mean-square deviation of 0.322 A between them. The O14 hydroxyls of the intercalated DOXs are within hydrogen bond distances to the O2P atoms of the A2p(aC3) and A8p(AC9) steps. The O2'-hydroxyl group from araC does not affect the binding of DAU-DOX or the conformation of the drug-DNA complexes. The results suggest that three major drug modifications on DNA, i.e., intercalation, covalent bond formation, and nucleoside analog incorporation, can coexist in the same DNA molecule without difficulty. When they occur in close proximity in DNA, they may provide an additive inhibitory effect for the target enzymes.

Base Sequence↗
PubMed1993

Structural influence of RNA incorporation in DNA: quantitative nuclear magnetic resonance refinement of d(CG)r(CG)d(CG) and d(CG)r(C)d(TAGCG).

RNA and DNA adopt different types of conformations, i.e., A-type with C3'-endo sugar pucker for RNA and B-type with C2'-endo sugar pucker for DNA, respectively. The structural influence of the incorporation of RNA nucleotides into DNA is less understood. In this paper, we present the three-dimensional structures of two RNA-containing oligonucleotides, d(CG)r(CG)d(CG) and d(CG)r(C)d-(TAGCG), as determined by the NMR refinement procedure, and assess the possible structural perturbation of DNA induced by RNA. With a single RNA insertion into an octamer DNA, its overall conformation remains as the canonical B-DNA, except that the sugar pucker of the rC3 residue is C3'-endo (pseudorotation angle P = 3.6 degrees). In contrast, the hybrid hexamer is neither the pure B-DNA nor the pure A-DNA conformation. Instead, we propose a model in which the DNA parts adopt B conformation, whereas the RNA part adopts A conformation, with the overall conformation closer to A-DNA. To ensure an exhaustive search of the conformational space, the model was subjected to 100-ps simulated annealing with slow cooling or 100-ps molecular dynamics with subsequent quenching. Models obtained at different time points of the trajectories were further subjected to the SPEDREF NOE refinement [Robinson & Wang (1992) Biochemistry 31, 3524] and they appeared to arrive at a convergent model (< 0.5 A RMSD for the central four base pairs). The consensus hexamer structure contains a significant discontinuity at the (rG4)p(dC5) step with a base pair tilt angle of 6.7 degrees and roll angle of 11.5 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold Temperature↗
PubMed1993

Iodonium ion-assisted synthesis of a haptenic tetrasaccharide fragment corresponding to the inner cell-wall glycopeptidolipid of Mycobacterium avium serotype 4.

Condensation of ethyl 2,4-di-O-benzoyl-1-thio-alpha-L-rhamnopyranoside with ethyl 3-O-benzyl-4-O-chloroacetyl-2-O-methyl-1-thio-beta-L-fucopyranoside in the presence of iodonium di-sym-collidine perchlorate afforded exclusively ethyl 2,4-di-O-benzoyl-3-O-(3-O-benzyl-4-O- chloroacetyl-2-O-methyl-alpha-L-fucopyranosyl)-1-thio-alpha-L-rhamnop yra noside. This disaccharide derivative was extended at C-1 with 3-benzyloxycarbonylaminopropyl 6-deoxy-3,4-O-isopropylidene-alpha-L- talopyranoside, using N-iodosuccinimide and triflic acid as the catalyst, to furnish 3-benzyloxycarbonylaminopropyl 6-deoxy-2-O-[2,4-di-O-benzoyl-3-O-(3-O-benzyl-4-O-chloroacetyl-2-O-me thy l- alpha-L-fucopyranosyl)-alpha-L-rhamnopyranosyl]-3,4-O-isopropylidene-alp ha-L- talopyranoside (20). Selective removal of the chloroacetyl group from 20, followed by condensation with ethyl 2,3-di-O-benzoyl-4-O-methyl-1-thio-alpha-L- rhamnopyranoside in the presence of the same thiophilic promoter, yielded a fully protected tetrasaccharide derivative. Deprotection of the latter gave the target compound 3-aminopropyl 6-deoxy-2-O-[3-O-[2-O- methyl-(4-O-methyl-alpha-L-rhamnopyranosyl)-alpha-L-fucopyranosyl]-alpha -L- rhamnopyranosyl]-alpha-L-talopyranoside (1).

Carbohydrate Sequence↗
PubMed1993

Verification of the position of the phosphate group in some synthetic phosphopeptides by fast-atom bombardment and tandem mass spectrometry.

Some synthetically obtained linear and cyclic phosphopeptides of low molecular weight have been studied by fast-atom bombardment and tandem mass spectrometry to verify the position of the phosphate group in these compounds. Based upon the occurrence/non-occurrence of loss of phosphoric acid from low abundance fragment ions induced by low- and high-energy collisions with target gases, it is shown that the position of the phosphate group in the phosphopeptides studied can be determined unequivocally.

Amino Acid Sequence↗
PubMed1993

Protein-conjugated synthetic di- and trisaccharides of pneumococcal type 17F exhibit a different immunogenicity and antigenicity than tetrasaccharide.

Overlapping synthetic disaccharide, trisaccharide and tetrasaccharide, derived from pneumococcal polysaccharide type 17F (PS17F), were coupled to keyhole limpet haemocyanin (KLH). The conjugates were tested in mice. The disaccharide-KLH and especially trisaccharide-KLH, in combination with Quil A, induced high titres of high-avidity anti-PS17F IgG. Both conjugates protected mice against challenge with Streptococcus pneumoniae 17F. Tetrasaccharide-KLH, although able to elicit anti-tetrasaccharide antibodies, induced a minimal non-protective anti-PS17F IgG response of low avidity. The tetrasaccharide-KLH conjugate, in contrast to the other conjugates, failed to bind rabbit anti-PS17F IgG.

Animals↗
PubMed1992

Structural consequences of a carcinogenic alkylation lesion on DNA: effect of O6-ethylguanine on the molecular structure of the d(CGC[e6G]AATTCGCG)-netropsin complex.

Exposure of cells to alkylating agents produces DNA lesions, most of which are repaired. However some alkyl lesions persist and play a role in inducing point mutations and the subsequent carcinogenic conversion. O6-Ethylguanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to N-ethyl-N-nitrosourea. We study the consequence of the e6G incorporation in DNA by X-ray crystallography. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) and the unmodified d(CGCGAATTCGCG), complexed to the minor groove binding drug netropsin. The space group of both crystals is P2(1)2(1)2(1), isomorphous to other related dodecamer DNA crystals. The structures have been solved by the molecular replacement method and refined by the constrained least-squares procedure to R-factors of approximately 16% at resolution of approximately 2.5 A. The two independent e6G-C base pairs in the DNA duplex adopt different base-pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with one three-centered hydrogen bond pair and one direct hydrogen bond between e6G4 and C21. In contrast, the e6G16-C9 base pair adopts a wobble configuration. The ethyl group is in the proximal orientation (to N7) in both base pairs. These observations enrich and support those found in the crystal structure of d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 and Hoechst 33342 [Sriram et al. (1992) EMBO J. 11, 225-232]. We suggest that a dynamic equilibrium between these two configurations for the e6G-C base pair is likely and would present an ambiguous signal to the cellular transcription, replication, or repair mechanisms. In contrast, thymine can pair with e6G in only one way, albeit imperfect, mimicking a Watson-Crick base pair. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication. In addition, we analyze the influence of the alkylation lesion on DNA and the molecular details of netropsin-DNA interaction. In the present two new netropsin complexes, the netropsin spans across five base pairs (starting halfway between C3-G22 and e6G4-C21 base pairs and ending at T8-A17 base pair) in the narrow minor groove. This is in contrast to the earlier crystal structure of netropsin complexed with another DNA dodecamer having the same AATT central core sequence, d(CGCGAATT[br5C]GCG) [Kopka et al. (1985) J. Mol. Biol. 272, 390-395]. In the latter structure, the netropsin lies between G4-br5C21 and br5C9-G16 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylation↗
PubMed1992

Unusual DNA conformation at low pH revealed by NMR: parallel-stranded DNA duplex with homo base pairs.

We have investigated the conformational potentials of several DNA oligonucleotides containing sequences related to 5'-CGA in neutral pH and low pH (< 5.0) conditions. One-dimensional proton NMR spectra show that d(CGATCG), d(TCGATCGA), and d(CGATCGATCG) exhibit new sets of resonances at low pH (approximately 3.8-4.4), when compared to those from the neutral pH samples. The low pH form and the neutral pH form are in slow equilibrium. Analyses of the data suggest that these sequences under low pH conditions adopt structures distinct from B-DNA. Two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY) data from the DNA hexamer d(CGATCG) of the neutral and low pH samples were used to analyze their respective structures in solution. An iterative NOE spectral-driven refinement procedure, SPEDREF [Robinson, H., & Wang, A. H.-J. (1992) Biochemistry 31, 3524-3533], was used to show that the neutral pH structure is close to canonical B-DNA. In contrast, analysis of the low pH form using the 2D NOESY data suggests that its structure is consistent with a right-handed parallel-stranded (PS) double helix with symmetrical non-Watson-Crick (C+:C, G:G, A:A, T:T) homo base pairs. Supporting evidence for the PS helix includes the asymmetric inversion-recovery relaxation times associated with the two ends of the helix. The structure is favored by the 5'-CGA sequence in which the cytosines provide the C+:C pairing for the nucleation step and the GpA step is significantly stabilized by the interstrand G-A stacking interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Synthesis of racemic 5-phosphonate analogues of myo-inositol 1,4,5-tris- and 1,3,4,5-tetrakis-phosphate.

(+/-)-2,3,6-Tri-O-benzyl-5-O-p-methoxybenzyl-myo-inositol and (+/-)-2,6-di-O-benzyl-5-O-p-methoxy-benzyl-myo-inositol, accessible readily from (+/-)-3,6-di-O-allyl-1,2-O-cyclohexylidene-myo-inositol, were phosphitylated with dibenzyl N,N-di-isopropylphosphoramidite, and the resulting phosphite triesters were oxidised with tert-butyl hydroperoxide to give the corresponding fully protected myo-inositol 1,4-bis- (12) and 1,3,4-tris-phosphate (13) derivatives. Cleavage of the p-methoxybenzyl group from 12 and 13, phosphonylation with bis[6-(trifluoromethyl)benzotriazol-1-yl] methylphosphonate or (difluoromethyl)phosphonic di(1,2,4-triazolide), followed by treatment in situ with benzyl alcohol, and then hydrogenolysis of the benzyl groups gave the 5-methylphosphonate and 5-[(difluoromethyl)phosphonate] analogues of myo-inositol 1,4,5-tris- and 1,3,4,5-tetrakis-phosphate. The 5-methylphosphonate analogue of myo-inositol 1,4,5-trisphosphate acted as a calcium antagonist in permeabilized human platelets.

Blood Platelets↗
PubMed1992

Heteronuclear NMR of DNA with the heteronucleus in natural abundance: facilitated assignment and extraction of coupling constants.

Two heteronuclear proton-carbon NMR experiments are applied to the DNA-octamer d(TTGGCCAA)2 with carbon in natural abundance. They lead to a complete assignment of the carbon resonances of the sugars and bases. In addition, several heteronuclear coupling constants, proton-carbon as well as proton-phosphorous and phosphorous-carbon, were determined. The information can be obtained in a reasonable measuring time and offers valuable information for a detailed picture of DNA structure.

Base Sequence↗
PubMed1992

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Selective and potent inhibition of different hepatic UDP-glucuronosyltransferase activities by omega,omega,omega-triphenylalcohols and UDP derivatives.

A homologous series of omega,omega,omega-triphenylalcohols and corresponding omega,omega,omega-triphenylalkyl-UDP derivatives was synthesized and tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes, with 1-naphthol, testosterone and bilirubin as substrates. Introduction of the UDP moiety in the triphenylalcohols increased their inhibition potency markedly toward the isoforms which glucuronidate 1-naphthol and testosterone, but strongly decreased that toward bilirubin. The inhibiting potency of the UDP-derivatives increased as a function of the length of the hydrocarbon chain. The best inhibitor 7,7,7-triphenylheptyl-UDP showed an I50 of 30 and 10 microM for 1-naphthol and testosterone glucuronidation, respectively; even a 1 mM concentration of the compound had little, if any, effect on bilirubin glucuronidation. The inhibition by 7,7,7-triphenylheptyl-UDP was mixed-type toward 1-naphthol, and non competitive toward testosterone (apparent K(i) 30 microM and 1.7 microM, respectively); on the other hand, the inhibition was competitive toward the common substrate UDP-glucuronic acid (apparent K(i) 1.9-1.2 microM). In addition, 7,7,7-triphenylheptyl-UDP (0.25-0.50 mM) almost inhibited glucuronidation of 1-naphthol and testosterone catalyzed by the recombinant rat liver UGT-2B1 and human liver UGT-1A1, whose cDNA has been expressed in V79 cells. In conclusion, the data indicate that 7,7,7-triphenyheptyl-UDP interacted competitively with the UDP binding site of UGT. The results also indicate that it is possible to design transition state analogue inhibitors with specificity for different UGT forms.

Alcohols↗