Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases. An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions. These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination.     

A note on auxotrophic mutants of cowpea rhizobia

We have examined a variety of common mutagens in producing auxotrophic mutants in cowpea rhizobia strains JRC23 and IRC256. While NTG (N-methyl-N-nitro-N-nitrosoguanidine), EMS (ethylmethane sulfonate), NA (nitrous acid), and UV (ultraviolet) irradiation were mutagenic with the strain JRC23, these mutagenic agents did not mutate strain IRC256. On the contrary, transposon mutagenesis with Tn5 yielded auxotrophs in strain IRC256 but not in strain JRC23, while only methionine (Met^–) auxotrophs from strain JRC23, histidine (His^–), and adenine plus thiamine (Ade^–+Thi^–) auxotrophs from strain IRC256 were isolated.     

Purine uptake by Alcaligenes eutrophus H16

The uptake of adenine, guanine, xanthine, hypoxanthine and uric acid by whole cells was studied, using spectrophotometric techniques, ¹⁴C-labelled compounds and metabolic inhibitors. Three different non-constitutive systems were shown to maintain the uptake of adenine and that of the pairs guanine/hypoxanthine and xanthine/uric acid. —Active transport of adenine was induced by adenine only, but passive uptake was also involved. Maximum K _T values of 110–131 M were observed at the pH optimum of 8.0. —Guanine and hypoxanthine were translocated by one single mechanism as indicated by K _T and K _I values. This system was induced by both these substances but its affinity was 51/2-times higher for guanine than for hypoxanthine; it was noncompetitively stimulated by Mg²⁺. — A further system, induced by xanthine and uric acid, catalyzed the uptake of both these compounds. It exhibited two pH optima (at pH 6.6 and 7.9); inactivation by heat and stimulation or inhibition by several compounds indicated that two separate mechanisms might be involved in the uptake of xanthine and uric acid.     

Formation of anhydrosugars in the chemical depolymerization of heparin.

In the reactions used to break heparin down to mono- and oligosaccharides, androsugars are formed at two stages. The first of these is the well-known cleavage of heparin with nitrous acid to convert the N-sulfated D-glucosamines to anhydro-D-mannose residues; this reaction has been studied in detail. It is demonstrated here that only low pH (less than 2.5) reaction conditions favor the deamination of N-sulfated D-glucosamine residues; the reaction proceeds very slowly at pH 3.5 or above. On the other hand, N-unsubstituted amino sugars are deaminated at a maximum rate at pH 4 with markedly reduced rates at pH2 or pH6. At room temperature solutions of nitrous acid lose one-fourth to one-third of their capacity to deaminate amino sugars in 1 h at all pHs. A low pH nitrous acid reagent which will convert heparin quantitatively to its deamination products in 10 min at room temperature is described, and a comparison of the effectiveness of this reagent with other commonly used nitrous acid reagents is presented. It is also shown that conditions used for acid hydrolysis of heparin convert approximately one-fourth of the L-iduronosyluronic acid 2-sulfate residues to a 2,5-anhydrouronic acid. This product is an artifact of the reaction conditions, and its formation represents one of several pathways followed in the acid-catalyzed cleavage of the glycosidic bond of the sulfated L-idosyluronic acid residues.     

The influence of formaldehyde fixation time on the rate of nitrous acid deamination of erythrocytes and spinal cord proteins

Summary Alcohol fixed blood films and fresh blocks of spinal cord were immersed in phosphate buffered neutral 10% formol for graded intervals, the films for 10, 30 min, 1, 2, 4, 8, 24 hr; the blocks for 2, 4, 6, 24 hr at 3 and 24° C; 1, 3, 7, 14, 21, 28, 42, 56 da, 3 and 14 mo at 24–26°. Graded deaminations in 2 N NaNO₂/HAc at 3° C were applied: 1, 2, 5, 10, 20, 30 min; 1, 2, 4, 6, 8, 12, 18, 24, 36 hr. Blood films were stained at pH 6 and 6.5, tissue at pH 4.5 and 5.0, both in azure A eosin B. The point at which erythrocytes reached a slightly bluish green was taken as the end point, since no further color change occurred on further exposure and erythrocytes were the last of usually deamination susceptible tissue elements to lose their oxyphilia on deamination. Deamination of alcohol fixed blood films is completed in about 2 min, of sublimate fixed spinal cord in about 1 hr. Progressive formaldehyde exposure increased deamination time of blood films to 10–20 min in 1 hr, to 6–8 hr in 4 hr and to 12 hr in 24 hr. The tissue deamination showed similar progressive increase of deamination time, slower with 3° C fixation than with 24–26°, reaching 18–36 hr by about 3 days formol, and remaining about the same thereafter.Supported by National Cancer Institute Grant No. C-04816, National Institutes of Health.     

Gamma Study of pH, Nitrite, and Salt Inhibition of Aeromonas hydrophila

The gamma hypothesis states that there are no interactions between antimicrobial environmental factors. The time to growth of Aeromonas hydrophila challenged with pH, NaNO₂, and salt combinations at 30°C was investigated. Data were examined using a model based on the gamma hypothesis (the gamma model), which takes into account variance-stabilizing transformations and which gives biologically relevant parameters. At high concentrations of NaNO₂ and at pHs of >6.0, the antimicrobial action of the nitrite ion has a strong influence (MIC = 2,033 mg liter⁻¹), whereas at pHs of <6, nitrous acid is dominant (MIC = 1.5 mg liter⁻¹). This change is not due to a “synergy” between pH and the nitrite ion but is due to the shift in the equilibrium concentrations of nitrous acid and nitrite in solution caused by pH. In combination with salt, the parameters found for the action of Na nitrite were identical to those found when it was examined in isolation. Therefore, pH, NaNO₂, and salt act independently on the growth of A. hydrophila. By expanding the gamma model with a cardinal temperature model, the results of fitting the model of Palumbo et al. (J. Food Prot. 54:429-435, 1994) to randomly produced environmental conditions could be reproduced, suggesting that temperature also has an independent effect.     

Purification and partial characterisation of a broad-range L-amino acid oxidase from Bacillus carotarum 2Pfa isolated from soil

The L-amino acid oxidase (L-aao) from Bacillus carotarum 2Pfa was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from crude sonicated cell extract by a combination of anion exchange chromatography and gel filtration. The purified enzyme was a dimer with a native relative molecular mass of approximately 102,000 to 115,000 and comprised two identical subunits of 54,000. The isoelectric point of the L-aao was at pH 4.8 the ph optimum was at 8.0–8.5 and the temperature optimum was at approximately 50° C. It was stable for several months at + 4° C and at –20° C. The enzyme contained 2 mol flavin adenine dinucleotide (FAD)/mol enzyme and exhibited relatively broad range substrate specificity, oxidising a total of ten L-amino acids and , albeit to a much lesser extent, seven D-amino acids. Kinetic studies revealed that the three aromatic L-amino acids were the preferred substrates.     

Effect of medium acidity on growth and rooting of different plant species growing in vitro

Micropropagated Choisya, Daphne, Delphinium, Hemerocallis, Hosta, Iris and Photinia were found to adjust the pH of Murashige and Skoog's plant tissue culture medium (initial pH 5.6 or 3.5) to different values depending on the species. When plant growth and rooting rates were determined after plants had been grown on media initially adjusted or buffered to values between 2.6 and 5.7 the different plant species were also found to have distinct pH requirements for optimal growth and/or rooting rates.Abbreviations MS Murashige & Skoog's (1962) medium - MS19 MS with additionally 10 g l^–1 sucrose - 80 mg l^–1 adenine sulphate and 130.9 mg l^–1 NaH₂PO₄ - BA 6-benzyladenine - NAA 1-naphthyl-acetic acid - IBA 3-indole-butyric acid - IAA 3-indole-acetic acid - 2iP N₆(2-isopentyl) adenine     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

Stability of 2'-deoxyxanthosine in DNA

The deamination of nucleobases in DNA occurs by a variety of mechanisms and results in the formation of hypoxanthine from adenine, uracil from cytosine, and xanthine and oxanine from guanine. 2′-Deoxyxanthosine (dX) has been assumed to be an unstable lesion in cells, yet no study has been performed under biological conditions. We now report that dX is a relatively stable lesion at pH 7, 37°C and 110 mM ionic strength, with a half-life (t_1/2) of 2.4 years in double-stranded DNA. The stability of dX as a 2′-deoxynucleoside (t_1/2 = 3.7 min at pH 2; 1104 h at pH 6) was increased substantially upon incorporation into a single-stranded oligodeoxynucleotide, in which the half-life of dX at different pH values was found to range from 7.7 h at pH 2 to 17 700 h at pH 7. Incorporation of dX into a double-stranded oligodeoxynucleotide resulted in a statistically insignificant increase in the half-life to 20 900 h at pH 7. Data for the pH dependence of the stability of dX in single-stranded DNA were used to determine the rate constants for the acid-catalyzed (2.6 × 10^–5 s^–1) and pH-independent (1.4 × 10^–8 s^–1) depurination reactions for dX as well as the dissociation constant for the N⁷ position of dX (6.1 × 10^–4 M). We conclude that dX is a relatively stable lesion that could play a role in deamination-induced mutagenesis.     

Hydrolysis of RNA monomers by extracts of Aspergillus niger NRRL3

Extracts of Aspergillus niger NRRL₃ catalyzed dephosphorylation of AMP, GMP, CMP and UMP over a wide range of pH values from pH 1.5 to pH 10. They also catalyzed hydrolytic deamination of only cytidine out of the tested ribonucleotides, ribonucleosides and bases. Neither cleavage of the N-glycosidic linkages of these nucleotides nor those of the corresponding nucleosides could be effected by the extracts. Phosphate liberation from the four RNA monomers seemed to be effected by two phosphate-non repressible phosphatases, acid and alkaline. Optimum activity of the acid phosphatase with all the substrates was at pH2 and 40 °C while that of the alkaline phosphatase was at pH8 and 40 °– 70 °C. Affinities of both phosphatases for the different ribonucleotides were in the order of magnitude AMP, CMP and phph > GMP > UMP. Freezing and thawing of the extracts had no effect either on the activities of two phosphatases or on that of the aminohydrolase. However, heating the extracts at 55° for 25 min, in absence of the substrate, inactivated the phosphatases and had no effect on the deaminase. No evidence for the involvement of specific nucleotidases in ribonucleotides dephosphorylation was recorded.     

Luminal alkalinization in the intestine of the goby

Summary The rate of luminal alkalinization in vitro byGillichthys mirabilis posterior intestine as measured by a manual pH stat technique was 0.70±0.05 Equiv/cm² h; acidification of the mucosal medium was never observed. The rate of HCO ₃ ^– secretion (J ^HCO ₃) was reduced by ouabain, serosally-applied DIDS, removal of serosal HCO ₃ ^– and replacement of media Cl^– with gluconate. HCO ₃ ^– secretion was enhanced replacement of Cl^– with isethionate and unaffected by mucosal DIDS, furosemide or acetazolamide.J ^HCO ₃ was reduced at mucosal pH above or below 7.5. These results support active HCO ₃ ^– secretion via a Cl^–/HCO ₃ ^– exchange mechanism on the basolateral membrane and a conductive exit pathway for HCO ₃ ^– , H⁺ or OH^– on the apical membrane.Abbreviations DIDS diisothiocyanostilbene-2,2-disulfonic acid - TEP transepithelial potential - GBR Gillichthyts bicarbonate Ringer - GUR Gillichthys unbuffered, bicarbonate-free Ringer - GER Gillichthys EPPS-buffered, bicarbonate-free Ringer - EPPS N-(2-hydroxyethyl)piperazine-N-3-propanesulfonic acid     

Nitrous acid damage to duplex deoxyribonucleic acid: distinction between deamination of cytosine residues and a novel mutational lesion.

The rate of nitrous acid deamination of labeled cytosine residues in native Escherichia coli deoxyribonucleic acid was monitored in vitro by release of acid-soluble counts after treatment with uracil deoxyribonucleic acid glycosylase. The reaction exhibited a lag and was not stimulate by several agents previously shown to enhance base substitution mutagenesis during nitrous acid treatment of duplex deoxyribonucleic acid. We conclude that a significant proportion of nitrous acid induced mutagenic lesions are novel lesions and not cytosine deaminations.     

Adaptation of Denitrifying Populations to Low Soil pH

Natural denitrification rates and activities of denitrifying enzymes were measured in an agricultural soil which had a 20-year past history of low pH (pH ca. 4) due to fertilization with acid-generating ammonium salts. The soil adjacent to this site had been limed and had a pH of ca. 6.0. Natural denitrification rates of these areas were of similar magnitude: 158 ng of N g⁻¹ of soil day⁻¹ for the acid soil and 390 ng of N g⁻¹ of soil day⁻¹ at the neutral site. Estimates of in situ denitrifying enzyme activity were higher in the neutral soil, but substantial enzyme activity was also detected in the acid soil. Rates of nitrous oxide reduction were very low, even when NO₃⁻ and NO₂⁻ were undetectable, and were ca. 400 times lower than the rates of N₂O production from NO₃⁻. Denitrification rates measured in slurries of the acid and neutral soil showed distinctly different pH optima (pH 3.9 and pH 6.3) which were near the pH values of the two soils. This suggests that an acid-tolerant denitrifying population had been selected during the 20-year period of low pH.     

Nitrogen transformations and nitrous oxide flux in a tropical deciduous forest in México

Summary Emissions of nitrous oxide and soil nitrogen pools and transformations were measured over an annual cycle in two forests and one pasture in tropical deciduous forest near Chamela, México. Nitrous oxide flux was moderately high (0.5–2.5 ng cm^–2 h^–1) during the wet season and low (<0.3 ng cm^–2 h^–1) during the dry season. Annual emissions of nitrogen as nitrous oxide were calculated to be 0.5–0.7 kg ha^–1 y^–1, with no substantial difference between the forests and pasture. Wetting of dry soil caused a large but short-lived pulse of N₂O flux that accounted for <2% of annual flux. Variation in soil water through the season was the primary controlling factor for pool sizes of ammonium and nitrate, nitrogen transformations, and N₂O flux.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

Disruption of sulfur cycling and acid neutralization in lakes at low pH

Experimental acidification of a softwater lake to below pH 5 fundamentally changed the sulfur cycle and lowered internal alkalinity generation (IAG). Prior to reaching pH 4.5, the balance of sulfur reduction and oxidation reactions within the lake was in favour of reduction, and the lake was a net sink for sulfate. In the four years at pH 4.5 the balance of reduction and oxidation reactions was in favour of oxidation, and there was a net production of sulfate (SO₄ ^2–) within the lake. Evidence indicating a decrease in net SO₄ ^2– reduction at pH 4.5 was also obtained in an anthropogenically acidified lake that had been acidified for many decades. In both lakes, the decrease in net SO₄ ^2– reduction appeared to be linked not to a simple inhibition of SO₄ ^2– reduction but rather to changes in benthic ecosystem structure, especially the development of metaphytic filamentous green algae, which altered the balance between SO₄ ^2– reduction and sulfur oxidation.At pH's above 4.5, net SO₄ ^2– reduction was the major contributor to IAG in the experimental lake, as it is in many previously studied lakes at pH 5 and above. At pH 4.5, the change in net annual SO₄ ^2– reduction (a decrease of 110%) resulted in a 38% decrease in total IAG. Because of the important role of net SO₄ ^2– reduction in acid neutralization in softwater lakes, models for predicting acidification and recovery of lakes may need to be modified for lakes acidified to pH <5.     

Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ

As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2-hydroxyethyl piperazine-N′-ethanesulfonic acid) and 37 C the K_m for N⁶-(Δ²-isopentenyl)adenine was 57.1 micromolar; N⁶-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the V_max for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N⁶-(Δ²-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism.     

Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN ⁶-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective K_i values were 3.5×10^–6 M and 9.5×10^–6 M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10^–4 M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers.     

Potential artifacts in the measurement of DNA deamination

Attack on DNA by some reactive nitrogen species results in deamination of adenine and guanine, leading to the formation of hypoxanthine and xanthine, respectively. Published levels of these products in cellular DNA have varied widely. Although these two deamination products are often measured by GC-MS analysis, the procedure of acid hydrolysis to release DNA bases for derivatization poses a risk of artifactual deamination of the DNA. In this study, we demonstrated the artifactual formation of these two deamination products during acid hydrolysis and hence developed a method for detecting and measuring 2'-deoxyinosine, the nucleoside of hypoxanthine. Our assay for 2'-deoxyinosine employs nuclease P1 and alkaline phosphatase to achieve release of the nucleosides from DNA, followed by HPLC prepurification with subsequent GC-MS analysis of the nucleosides. This assay detected an increase in the levels of 2'-deoxyinosine in DNA when commercial salmon testis DNA was treated with nitrous acid. We also used it to measure levels in various rat tissues of both normal and endotoxin-treated rats, but could not find increased 2'-deoxyinosine formation in tissues even though *NO production was substantially increased.