Preferential Modification Research Articles

Sequence Distribution of Acetaldehyde-Derived N2-Ethyl-dG Adducts along Duplex DNA

Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence-selective formation of other tobacco carcinogen-DNA adducts along K-ras- and p53-derived duplexes and the preferential modification of endogenously methylated CG dinucleotides by benzo[a]pyrene diol epoxide and acrolein.

DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites

The Dnd (DNA degradation) phenotype, reflecting a novel DNA modification by sulfur in Streptomyces lividans 1326, was strongly aggravated when one (dndB) of the five genes (dndABCDE) controlling it was mutated. Electrophoretic banding patterns of a plasmid (pHZ209), reflecting DNA degradation, displayed a clear change from a preferential modification site in strain 1326 to more random modifications in the mutant. Fourteen randomly modifiable sites on pHZ209 were localized, and each seemed to be able to be modified only once. Residues in a region (5′-c–cGGCCgccg-3′) including a highly conserved 4-bp central core (5′-GGCC-3′) in a well-documented preferential modification site were assessed for their necessity by site-directed mutagenesis. While the central core (GGCC) was found to be stringently required in 1326 and in the mutant, ‘gccg’ flanking its right could either abolish or reduce the modification frequency only in the mutant, and two separate nucleotides to the left had no dramatic effect. The lack of essentiality of DndB for S-modification suggests that it might only be required for enhancing or stabilizing the activity of a protein complex at the required preferential modification site, or resolving secondary structures flanking the modifiable site(s), known to constitute an obstacle for efficient modification.

Photo-Induced Structural Changes in Titanium Alkoxides for Directing Molecular Assembly

ABSTRACTThe effects of ultraviolet irradiation on a heteroleptic titanium alkoxide ((OPy)2Ti(4MP)2) have been investigated. The molecule has been studied in solution and in thin film form using FTIR and Raman spectroscopies. Quantum computational modeling was used to associate vibrational modes with structural moieties present in the molecule. In all cases examined, a preferential photoinduced modification in vibrational resonances linked to the 4-mercaptophenol (4MP) ligand was observed. In contrast, little or no change was exhibited in the vibrational structure of the OPy ligands.

Cu adsorption on carboxylic acid-terminated self-assembled monolayers: a high-resolution X-ray photoelectron spectroscopy study

The interaction between vacuum-deposited Cu and a carboxylic acid-terminated self-assembled monolayer (SAM) on Au(111) has been investigated using high-resolution X-ray photoelectron spectroscopy (XPS). Upon adsorption of 3-mercaptopropionic acid from the gas phase on Au(111), C 1s, O 1s, and S 2p line shapes and binding energies are consistent with the formation of a thiolate surface intermediate with an intact carboxylic acid function. Deposition of increasing amounts of Cu results in preferential modification of the hydroxyl group O 1s peak indicating unidentate complexation. Consistent with selective bonding, Cu induces changes in the carboxylic acid C 1s region with no evidence of reaction with the methylene or thiolate units. The Cu 2p core level is comparable with bulk, suggesting cluster growth and weak Cu-SAM interactions. The reactivity is nevertheless sufficient to prevent metal penetration to the Au substrate surface.

Numerical Analyses on Wall-Attaching Offset Jet with Various Turbulent $k-{\varepsilon}$ Models and Skew-Upwind Scheme

Four turbulent models (i.e., standard model, modified models with streamline curvature modification and/or preferential dissipation modification) are applied in order to analyze the turbulent flow of wall-attaching offset jet. For numerical convergence, this paper develops a method of slowly increasing the convective effect induced by skew-velocity in skew-upwind scheme (hereafter called Partial Skewupwind Scheme). Even though the method was simple, it was efficient in view of convergent speed, computer memory storage, programming, etc. The numerical results of all models show good prediction in first order calculations (i.e., reattachment length, mean velocity, pressure), while they show some deviations in ·second order (i.e., kinetic energy and its dissipation rate). Like the previous results obtained by upwind scheme, the streamline curvature modification results in better prediction, while the preferential dissipation modification does not.

Cytosine Methylation in a CpG Sequence Leads to Enhanced Reactivity with Benzo[a]pyrene Diol Epoxide That Correlates with a Conformational Change

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.

Catalytic DNA- and RNA-hydrolyzing antibodies from milk of healthy human mothers.

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab')2 and Fab fragments. Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of catalytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infections until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.

Circulating advanced glycation peptides in streptozotocin-induced diabetic rats: Evidence for preferential modification of IgG light chains

As the glycation/glycoxidation hypothesis for the genesis of diabetic complications is achieving widespread acceptance, much attention is being paid to the role of low molecular weight advanced glycation (AGE) adducts, as second generation glycating agents. We set out a study with the objective of attesting the presence of increased amounts of AGE-peptides in the circulation of streptozotocin-induced diabetic rats and to determine the nature of the plasma proteins which are main targets for advanced glycation. AGE (Ex 370/Em 440 nm) and pentosidine fluorescence ( Ex 335/Em 385 nm) were significantly higher in plasma from diabetic rats after only one month of hyperglycemia as compared to controls (35 ± 7 vs 25 ± 2 AU, p < 0.05 and 54 ± 14 vs 27 ± 3 AU, p < 0.01 respectively). AGE-peptides ( < 10 kDa) were more than two-fold higher in diabetic animals. Immunoblots after SDS-PAGE of plasma proteins showed that AGE-IgG displayed a selective predominant increment in the same animals. When native rat IgG was incubated in the presence of AGE-peptides isolated from diabetic animals, AGE modification was already apparent after only 24 h of incubation, and was particularly important for light chains. AGE-immunoreactive light chains displayed an apparent increase in molecular weight. Aminoguanidine prevented, while copper enhanced AGE binding to IgG light chains. Our data validate the streptozotocin-induced diabetic rat as a model reproducing the presence of circulating AGE-peptides, give evidence that IgG are preferential targets for advanced glycation in plasma and suggest that this modification, mediated by AGE-peptides, can be prevented by aminoguanidine.

Preferential Modification of Nuclear Proteins by a Novel Ubiquitin-like Molecule

Sentrin is a novel ubiquitin-like protein that protects cells against both anti-Fas and tumor necrosis factor-induced cell death. Antiserum recognizing the N terminus of sentrin revealed the presence of a 18-kDa sentrin monomer, a 90-kDa band (p90), and multiple high molecular mass bands. Because sentrin possesses the conserved Gly-Gly residues near the C terminus, it is likely that these additional bands represent conjugation of sentrin to other proteins in a manner that is similar to the ubiquitination pathway. Transient expression of hemagglutinin epitope-tagged sentrin mutants in COS cells demonstrated that the sentrin C terminus is cleaved, which allows it to be conjugated to other proteins via the conserved C-terminal Gly residue. Immunocytochemical staining and cell fractionation analysis demonstrated that sentrin monomer is localized predominantly to the cytosol. However, p90 and the majority of sentrinized proteins appeared to be localized to the nucleus. When the conserved Gly-Gly residues of sentrin were changed to Gly-Ala, only sentrin monomer and p90 but not the high molecular mass bands were observed. Thus, p90 generation appears to be required for the formation of high molecular mass bands in the nucleus. Taken together, sentrinization represents a novel pathway for nuclear protein modification, which is distinct from ubiquitination.

Cytosine methylation determines hot spots of DNA damage in the human P53 gene.

In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previously mapped the distribution of (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts along the human P53 gene [Denissenko, M. F., Pao, A., Tang, M.-s. & Pfeifer, G. P. (1996) Science 274, 430-432]. Strong and selective formation of adducts occurred at guanines in CpG sequences of codons 157, 248, and 273, which are the major mutational hot spots in lung cancer. Chromatin structure was not involved in preferential modification of these sites by BPDE. To investigate other possible mechanisms underlying the selectivity of BPDE binding, we have mapped the adducts in plasmid DNA containing genomic P53 sequences. The adduct profile obtained was different from that in genomic DNA. However, when cytosines at CpG sequences were converted to 5-methylcytosines by the CpG-specific methylase SssI and the DNA was subsequently treated with BPDE, adduct hot spots were created which were similar to those seen in genomic DNA where all CpGs are methylated. A strong positive effect of 5-methylcytosine on BPDE adduct formation at CpG sites was also documented with sequences of the PGK1 gene derived from an active or inactive human X chromosome and having differential methylation patterns. These results show that methylated CpG dinucleotides, in addition to being an endogenous promutagenic factor, may represent a preferential target for exogenous chemical carcinogens. The data open new avenues concerning the reasons that the majority of mutational hot spots in human genes are at CpGs.

Regioselective Hydrogen-atom Transfer from Triglycine†

C. J. Easton, J. B. Kelly and C. M. Ward, J. Chem. Res. (S), 1997, 470 DOI: 10.1039/A703000B

NUMERICAL STUDY OF TURBULENT FORCED CONVECTION IN A PERIODICALLY RIBBED CHANNEL WITH OSCILLATORY THROUGHFLOW

Engineering Systems International/Uihon ESI K.K., 2-47-18 Uehara, Shibuya. Tobo 151, Japan SUMMARY Numerical computations are performed on the fully developed flow and heat transfer in a periodically ribbed channel with oscillatory throughflow. A uniform heat flux is imposed at the lower plate of the channel. An externally sustained pressure gradient vanes sinusoidally in time. A low-turbulent-Reynolds-number version of the k+ two-equation model of turbulence is invoked, together with a preferential dissipation modification, to predict the complex turbulent flow field. Computed results indicate that much heat transfer enhancement is expected by increasing the Womersley number, which measures the relative strength of the oscillatory motion to the viscous effects. Turbulent heat transfer in a channel with rib-roughened walls is of much engineering relevance. It is known to be an effective means of heat transfer enhancement in compact heat exchangers, cooling of electronic devices and other types of thermal engineering equipment. A higher heat transfer rate may be expected by imposing on the channel an externally sustained oscillatory pressure gradient, by which better mixing of the medium can be accomplished. It is thought to be a promising technique for cooling high-density electronic circuit boards, for which an excessively high flow rate of the cooling agent is not desirable from a mechanical design point of view. Despite its potential for innovative engineering applications, the available information on turbulent heat transfer in oscillatory flow is limited. Ohmi et d.' measured the velocity profiles of oscillatory througMow in a rectangular duct using hot-wire anemometry. Instantaneous velocity profiles for turbulent oscillatory flow were found to be similar to those of non-oscillatory flow. Consequently, the profiles contained little phase shift, contrasting with their laminar flow counterparts. The same authors have also constructed a flow regime diagram, thereby identifying the role of the mechanically imposed frequency (characterized by the Womersley number) in the flow transition. Their experiments were conducted under an isothermal condition. Features of the flow field and mass transfer rate were investigated for a wavy walled channel by Nishimura et aL2 With a smoothly contoured wall, flow separation and subsequent growth of a vortex, which eventually occupied the entire flow passage, were clearly observable in the acceleration stage. In the deceleration stage accompanying the reversal in the flow direction, these vortices were

Mitogen-stimulated phosphorylation of histone H3 is targeted to a small hyperacetylation-sensitive fraction.

Diverse agents, including growth factors and phorbol esters, induce rapid transcriptional activation of a subset of immediate-early (IE) genes that include the protooncogenes c-fos and c-jun. Among the earliest nuclear signaling events concomitant with IE gene activation is the phosphorylation of nucleosomal histone H3 in its basically charged N-terminal tail. This highly conserved domain is also subject to reversible posttranslational acetylation at specific lysine residues, a process implicated in transcriptional regulation. We show here that H3 phosphorylation associated with G0-G1 transition affects only a small fraction of this histone in the nucleus. Moreover, this fraction is biochemically distinct from bulk H3 in being extremely sensitive to sodium butyrate-induced hyperacetylation. However, acetylation itself does not predispose H3 to phosphorylation, nor does phosphorylation predispose H3 to enhanced acetylation. Further, selectivity is not based on preferential modification of particular histone H3 subtypes. Thus, the mitogen-regulated kinase that phosphorylates histone H3 is restricted to a small subset of nucleosomes that is especially susceptible to hyperacetylation.

Elimination of atrioventricular nodal reentrant tachycardia using discrete slow potentials to guide application of radiofrequency energy.

Ablation of the slow pathway has been performed to eliminate atrioventricular (AV) nodal reentrant tachycardia (AVNRT) either by a surgical approach or by using radiofrequency catheter technique guided by retrograde slow pathway activation mapping. From previous experience of midseptal and posteroseptal mapping, we were aware of the existence of peculiar slow potentials in most humans. Postulating their role in AVNRT, we studied these potentials and the effects of radiofrequency energy. Sixty-four patients (mean age, 48 +/- 19 years) with the usual form of AVNRT were studied. Slow, low-amplitude potentials were recorded when using the anterograde AV conducting system. Slow potentials occupied all (giving a continuum of electrograms) or some of the time between the atrial and ventricular electrograms. Their most specific patterns were their progressive response to increasing atrial rates, which resulted in a dramatic decline in amplitude and slope, a corresponding increase in duration, and a separation from preceding atrial potentials until the disappearance of any consistent activity. Slow potentials were recorded along a vertical band at the mid or posterior part of the septum near the tricuspid annulus. Radiofrequency energy applied at the slow potential site resulted in interruption of induced tachycardia within a few seconds and rendered tachycardia noninducible in all patients. A median of two impulses was delivered to each patient. In 69% of patients, postablation atrial stimulation cannot achieve a long atrial-His interval, which previously was critical for tachycardia induction or maintenance. No patient had AVNRT over a follow-up period of 1-16 months, and all had preserved AV conduction. In all except two patients, the PR interval was unchanged. In 47 patients, long-term electrophysiological studies confirmed the efficacy of ablation and the nonreversibility of results by isoproterenol; however, echo beats remained inducible in 40% of patients. An area showing slow potentials is present at the perinodal region in humans. In patients with AVNRT, application of radiofrequency energy renders tachycardia noninducible through the preferential modification of the anterograde slow pathway. With present clinical methods, the exact origin and significance of these physiological potentials cannot be specified.

Interaction of RNA polymerase II with acetylated nucleosomal core particles

Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery.

Preferential modification of guanine bases in DNA by dimethyldioxirane and its application to DNA sequencing

From gel sequencing experiments with 32P-end-labelled oligodeoxyribonucleotides, it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3′- or 5′-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA.

Nucleic acid binding and mutagenicity of active metabolites of 2-amino-3,8-dimethylimidazo[4,5-ƒ]quinoxaline

2-Amino-3,8-dimethylimidazo[4,5-ƒ]quinoxaline (MeIQx) is a potent mutagen and carcinogen present in heated foodstuffs. The covalent binding of MeIQx to calf thymus DNA and calf liver RNA with microsomal activation was demonstrated. A major metabolite which exerts a direct mutagenic effect on S. typhimurium TA98 was found by HPLC analysis after incubation of MeIQx with rat liver microsomal fraction. The metabolite was identified as 2-hydroxyamino-3,8-dimethylimidazol[4,5-ƒ]quinoxaline (N- OH-MeIQx). Synthetic N-OH-MeIQx was found to bind non-enzymatically to DNA and RNA at neutral pH even at 0°C. Addition of acetic anhydride increased the binding of N-OH-MeIQx to DNA 10 times. These results suggest that MeIQx is metabolized to N-OH-MeIQx by microsomal cytochrome P-450 and further activated to an acetylated form that binds efficiently to nucleic acids in rat liver. Preferential modification of polyguanylic acid suggests that guanine residues of DNA are mainly modified with MeIQx. Synthetic N-OH-MeIQx exerted direct mutagenic activity on S. typhimurium TA98 inducing 150 000 rev/μg. Pentachlorophenol (PCP) caused a dose-dependent inhibition of this mutagenic effect, but 2,6-dichloro-4-nitrophenol (DCNP) did not. Thus the acetyltransferase of S. typhimurium seems to be important for the high mutagenicity of MeIQx after its microsomal activation.

Preferential modification of GC boxes by benzo[a]pyrene‐7,8‐diol‐9,10‐epoxide

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.

Chemical modification of tumour blood flow.

The preferential modification of tumour blood flow with chemical agents has been extensively investigated for the purpose of improving the treatment of tumours with ionizing radiation, chemicals and hyperthermia. In this paper I have utilized representative vasoactive drugs to demonstrate the various mechanisms via which tumour haemodynamics can be modified selectively. Chemicals which alter tumour blood flow by changing blood viscosity are also discussed. Of particular interest are the calcium entry blockers, flunarizine and verapamil, which have been shown to increase tumour blood flow, tumour oxygenation, tumour radiation sensitivity and the cytotoxicity of chemotherapeutic agents. These encouraging research results provide compelling evidence that these two drugs may prove to be useful adjuvants for radiation therapy.

The metal-metal oxide interface: A study of thermally-activated diffusion at the Ni/Al 2O 3 interface using electron spectroscopies

The behavior of Ni atoms deposited on well-characterized Al2O3 films, prepared by oxidizing an atomically clean Al(111) surface, has been studied using high-resolution electron energy loss spectroscopy (EELS) and Auger electron spectroscopy (AES). At 200 K, the deposited Ni forms three-dimensional Ni clusters on the Al2O3/Al(111) substrate. Interaction of these Ni metal clusters with surface Al-O species is directly detected by observation of the preferential modification of surface phonon mode frequencies and intensities in contrast to the behavior of a bulk phonon mode of the Al2O3 substrate. Heating the Ni/Al2O3 deposit above 200 K gives rise to two thermally induced processes: Process, I, smoothing (below ∼400 K) of the three-dimensional Ni clusters and Process II, diffusion (400–700 K) of the Ni overlayer through channels in the Al2O3 film. The metallic aluminum beneath the Al2O3 film is found to provide a major driving force for the inward diffusion of the Ni overlayer by Ni-Al alloy formation. This work represents the first application of EELS to the study of transport processes in a thin film system.