Relatively small increases in the steady-state levels of nucleobase deamination products in DNA from human TK6 cells exposed to toxic levels of nitric oxide.
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Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 microM NO along with 186 microM O2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method, and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2'-deoxyuridine (dU) and 2'-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 microM NO and 186 microM O2 for 12 h (1260 microM x min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 h recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI, and dU, respectively, to reach the following levels: dX, 7 (+/- 1) per 10(6) nt; dI, 25 (+/- 2.1) per 10(6) nt; and dU, 40 (+/- 3.8) per 10(6) nt. dO was not detected above the limit of detection of 6 lesions per 10(7) nt in 50 microg of DNA. A 12 h exposure to 0.65 microM NO and 190 microM O2 (468 microM x min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a approximately 15% (+/- 3.6) reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA. (+info)
Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli.
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In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products. The purpose of this study was to determine which of these two agents is more responsible for endogenous nitrosative mutagenesis. An nfi (endonuclease V) mutant was grown anaerobically with nitrate or nitrite, conditions under which it has a high frequency of A:T-to-G:C transition mutations because of a defect in the repair of hypoxanthine (nitrosatively deaminated adenine) in DNA. These mutations could be greatly reduced by two means: (i) introduction of an nirB mutation, which affects the inducible cytoplasmic nitrite reductase, the major source of nitric oxide during nitrate or nitrite metabolism, or (ii) flushing the anaerobic culture with argon (which should purge it of nitric oxide) before it was exposed to air. The results suggest that nitrosative mutagenesis occurs during a shift from nitrate/nitrite-dependent respiration under hypoxic conditions to aerobic respiration, when accumulated nitric oxide reacts with oxygen to form endogenous nitrosating agents such as N2O3. In contrast, mutagenesis of nongrowing cells by nitrous acid was unaffected by an nirB mutation, suggesting that this mutagenesis is mediated by N2O3 that is formed directly by the condensation of nitrous acid. (+info)
Involvement of semicarbazide-sensitive amine oxidase-mediated deamination in lipopolysaccharide-induced pulmonary inflammation.
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Semicarbazide-sensitive amine oxidase (SSAO) resides on the vascular endothelium and smooth muscle cell surface and is capable of deaminating short chain aliphatic amines and producing toxic aldehydes and hydrogen peroxide. The enzyme, also known as a vascular adhesion protein-1, is involved in the inflammation process. This intriguing protein with dual functions is increased in the serum of diabetic and heart failure patients. In the present study we assessed the involvement of SSAO in a lipopolysaccharide-induced pulmonary inflammation model using transgenic mice that overexpress human vascular adhesion protein-1. Overexpression of SSAO activity increased the formation of protein-formaldehyde deposits in tissues. Lysine residues of proteins were the primary targets for cross-linkage with formaldehyde derived from deamination of methylamine. Lipo-polysaccharide-induced increases in inflammatory cells in the bronchoalveolar lavage (BAL) fluid were significantly higher in the transgenic than in the nontransgenic mice. BAL cell counts were also higher in the untreated transgenic than in nontransgenic mice. Blocking SSAO activity with a selective inhibitor significantly reduced the number of neutrophils as well as levels of macrophage inflammatory protein-1alpha, granulocyte colony-stimulating factor, tumor necrosis factor-alpha, and interleukin-6 in the BAL fluid. Inhalation of methylamine also increased BAL neutrophil counts. Together, these results suggest a role for SSAO-mediated deamination in pulmonary inflammation. (+info)
The transcription elongation complex directs activation-induced cytidine deaminase-mediated DNA deamination.
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Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter. (+info)
Probing nitrogen-sensitive steps in the free-radical-mediated deamination of amino alcohols by ethanolamine ammonia-lyase.
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The contribution of C-N bond-breaking/making steps to the rate of the free-radical-mediated deamination of vicinal amino alcohols by adenosylcobalamin-dependent ethanolamine ammonia-lyase has been investigated by 15N isotope effects (IE's) and by electron paramagnetic resonance (EPR) spectroscopy. 15N IE's were determined for three substrates, ethanolamine, (R)-2-aminopropanol, and (S)-2-aminopropanol, using isotope ratio mass spectrometry analysis of the product ammonia. Measurements with all three substrates gave measurable, normal 15N IE's; however, the IE of (S)-2-aminopropanol was approximately 5-fold greater than that of the other two. Reaction mixtures frozen during the steady state show that the 2-aminopropanols give EPR spectra characteristic of the initial substrate radical, whereas ethanolamine gives spectra consistent with a product-related radical (Warncke, K.; Schmidt, J. C.; Kee, S.-C. J. Am. Chem. Soc. 1999, 121, 10522-10528). The steady-state concentration of the radical with (R)-2-aminopropanol is about half that observed with the S isomer, and with (R)-2-aminopropanol, the steady-state level of the radical is further reduced upon deuteration at C1. The results show that relative heights of kinetic barriers differ among the three substrates such that levels or identities of steady-state intermediates differ. 15N-sensitive steps are significant contributors to V/K with (S)-2-aminopropanol. (+info)
Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI.
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Hydrolytic deamination of cytosines in DNA creates uracil and, if unrepaired, these lesions result in C to T mutations. We have suggested previously that a possible way in which cells may prevent or reduce this chemical reaction is through the binding of proteins to DNA. We use a genetic reversion assay to show that a restriction enzyme, PspGI, protects cytosines within its cognate site, 5'-CCWGG (W is A or T), against deamination under conditions where no DNA cleavage can occur. It decreases the rate of cytosine deamination to uracil by 7-fold. However, the same protein dramatically increases the rate of deaminations within the site 5'-CCSGG (S is C or G) by approximately 15-fold. Furthermore, a similar increase in cytosine deaminations is also seen with a catalytically inactive mutant of the enzyme showing that endonucleolytic ability of the protein is dispensable for its mutagenic action. The sequences of the mutants generated in the presence of PspGI show that only one of the cytosines in CCSGG is predominantly converted to thymine. Our results are consistent with PspGI 'sensitizing' the cytosine in the central base pair in CCSGG for deamination. Remarkably, PspGI sensitizes this base for damage despite its inability to form stable complexes at CCSGG sites. These results can be explained if the enzyme has a transient interaction with this sequence during which it flips the central cytosine out of the helix. This prediction was validated by modeling the structure of PspGI-DNA complex based on the structure of the related enzyme Ecl18kI which is known to cause base-flipping. (+info)
Group B streptococcal type II and III conjugate vaccines: physicochemical properties that influence immunogenicity.
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Recent efforts toward developing vaccines against group B streptococci (GBS) have focused on increasing the immunogenicity of GBS polysaccharides by conjugation to carrier proteins. However, partial depolymerization of GBS polysaccharides for the production of vaccines is a difficult task because of their acid-labile, antigenically critical sialic acids. Here we report a method for the partial depolymerization of type II and III polysaccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-d-mannose residues. Through the free aldehydes of their newly formed end groups, the fragments were conjugated to tetanus toxoid by reductive amination. The resulting conjugates stimulated the production in animals of high-titer type II- and III-specific antibodies which induced opsonophagocytic killing of type II and III strains of group B streptococci. For the type II conjugates, immunogenicity increased as oligosaccharide size decreased, whereas for type III conjugates, the size of the oligosaccharides did not significantly influence immunogenicity. When oligosaccharides of defined size were conjugated through sialic acid residues, the resulting cross-linkages were shown to affect immunogenicity. When oligosaccharides were conjugated through terminal aldehyde groups generated by deamination, modification of the exocyclic chain of sialic acid did not influence immunogenicity. (+info)
Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation.
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Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z(n)-57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments. (+info)