Multiple deficiencies in antioxidant enzymes in mice result in a compound increase in sensitivity to oxidative stress

To examine the effect of compound deficiencies in antioxidant defense, we have generated mice (Sod2^+/−/Gpx1^−/−) that are deficient in Mn superoxide dismutase (MnSOD) and glutathione peroxidase 1 (Gpx1) by breeding Sod2^+/− and Gpx1^−/− mice together. Although Sod2^+/−/Gpx1^−/− mice showed a 50% reduction in MnSOD and no detectable Gpx1 activity in either mitochondria or cytosol in all tissues, they were viable and appeared normal. Fibroblasts isolated from Sod2^+/−/Gpx1^−/− mice were more sensitive (4- to 6-fold) to oxidative stress (t-butyl hydroperoxide or γ irradiation) than fibroblasts from wild-type mice, and were twice as sensitive as cells from Sod2^+/− or Gpx1^−/− mice. Whole-animal studies demonstrated that survival of the Sod2^+/−/Gpx1^−/− mice in response to whole body γ irradiation or paraquat administration was also reduced compared with that of wild-type, Sod2^+/−, or Gpx1^−/− mice. Similarly, endogenous oxidative stress induced by cardiac ischemia/reperfusion injury led to greater apoptosis in heart tissue from the Sod2^+/−/Gpx1^−/− mice than in that from mice deficient in either MnSOD or Gpx1 alone. These data show that Sod2^+/−/Gpx1^−/− mice, deficient in two mitochondrial antioxidant enzymes, have significantly enhanced sensitivity to oxidative stress induced by exogenous insults and to endogenous oxidative stress compared with either wild-type mice or mice deficient in either MnSOD or Gpx1 alone.     

Effect of p53 genotype on gene expression profiles in murine liver

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the “guardian of the genome”. Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53^−/− and p53^+/− mice. Six male mice from each genotype (p53^+/+, p53^+/−, and p53^−/−) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53^+/+ and p53^+/− or between p53^+/+ and p53^−/− at the level of p ≤ 0.05. Both genes with increased expression and decreased expression were identified in p53^+/− and in p53^−/− mice. Most notable in the gene list derived from the p53^+/− mice was the significant reduction in p53 mRNA. In the p53^−/− mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.     

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk^+/+, Tk^+/− and Tk^−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk^+/+ and Tk^+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk^−/− mice. Unexpectedly, neonatal Tk^−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk^+/+ and Tk^+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk^−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk^+/+ mice). Additional assays performed on untreated Tk^−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk^−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk^−/− mice suggest the presence of an endogenous genotoxic activity in these mice.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

A new purification procedure and molecular properties of Pseudomonas cytochrome oxidase

A procedure has been developed for purification of the cytochrome oxidase from Pseudomonas aeruginosa (EC 1.9.3.2) using DEAE- and CM-cellulose chromatography, gel filtration and crystallization. The final preparation was found to be homogeneous according to ultracentrifugal and disc electrophoretic criteria. The crystalline preparation also exhibited nitrite reductase activity. The spectrum of the enzyme characterizes it as cytochrome cd. At 280 nm E^{1 %}_{1 cm} was 18.5 after dry weight analysis.

The molecular weight of the cytochrome oxidase was calculated to be 119000 based on a sedimentation coefficient s° _20,w = 7.36 S, diffusion coefficient D _20,w = 5.36×10⁻⁷ cm²×s⁻¹ and partial specific volume of 0.72 ml/g. The iron content of the enzyme (0.166 %) indicates that this entity contains four iron atoms per molecule. Succinylation of the enzyme produced two probably identical subunits containing both hemes c and d, having a sedimentation coefficient s° _20,w = 4.30 S and an approximate molecular weight of 65000. In dodecylsulphate-acrylamide gel electrophoresis the cytochrome oxidase also dissociates into two subunits with molecular weight of 63000.     

Electrospray mass spectrometry of trans-[Ru(NO)Cl(dpaH)2] (dpaH=2,2′-dipyridylamine) and ‘caged NO’, [RuCl3(NO)(H2O)2]: loss of HCl and NO from positive ions versus NO and Cl from negative ions

The positive ion electrospray mass spectrometry (ESI-MS) of trans-[Ru(NO)Cl)(dpaH)₂]Cl₂ (dpaH=2,2′-dipyridylamine), obtained from the carrier solvent of H₂O–CH₃OH (50:50), revealed 1+ ions of the formulas [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ (m/z=508), [Ru^IIICl(dpaH)(dpa⁻)]⁺ (m/z=478), [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472), [Ru^III(dpa)₂]⁺ (m/z=442), originating from proton dissociation from the parent [Ru^II(NO⁺)Cl(dpaH)₂]²⁺ ion with subsequent loss of NO (17.4% of dissociative events) or loss of HCl (82.6% of dissociative events). Further loss of NO from the m/z=472 fragment yields the m/z=442 fragment. Thus, ionization of the NH moiety of dpaH is a significant factor in controlling the net ionic charge in the gas phase, and allowing preferential dissociation of HCl in the fragmentation processes. With NaCl added, an ion pair, {Na[Ru^II(NO)Cl(dpa)₂]}⁺ (m/z=530; 532), is detectable. All these positive mass peaks that contain Ru carry a signature ‘handprint’ of adjacent m/z peaks due to the isotopic distribution of ¹⁰⁴Ru, ¹⁰²Ru, ¹⁰¹Ru, ⁹⁹Ru, ⁹⁸Ru and ⁹⁶Ru mass centered around ¹⁰¹Ru for each fragment, and have been matched to the theoretical isotopic distribution for each set of peaks centered on the main isotope peak. When the starting complex is allowed to undergo aquation for two weeks in H₂O, loss of the axial Cl⁻ is shown by the approximately 77% attenuation of the [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ ion, being replaced by the [Ru^II(NO⁺)(H₂O)(dpa)₂]⁺ (m/z=490) as the most abundant high-mass species. Loss of H₂O is observed to form [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472). No positive ion mass spectral peaks were observed for RuCl₃(NO)(H₂O)₂, ‘caged NO’. Negative ions were observed by proton dissociation forming [Ru^II(NO)Cl₃(H₂O)(OH)]⁻ in the ionization chamber, detecting the parent 1− ion at m/z=274, followed by the loss of NO as the main dissociative pathway that produces [Ru^IIICl₃(H₂O)(OH)]⁻ (m/z=244). This species undergoes reductive elimination of a chlorine atom, forming [Ru^IICl₂(H₂O)(OH)]⁻ (m/z=208). The ease of the NO dissociation is increased for the negative ions, which should be more able to stabilize a Ru^III product upon NO loss.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺ sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli.

We have characterized 202 lacI⁻ mutations, and 158 dominant lacI^−d mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The −(G:C) frameshifts were the dominant mutational class in the lacI⁻ collections of both NR6112 and EE125, and in the lacI^−d collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI^−d collection. This study completes a comprehensive analysis of 1194 lacI⁻ and 348 lacI^−d mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

Metal ion substitution in phospholipase C (Bacillus cereus) and its effect on enzyme stability

The effect of guanidinium chloride solutions on the circular dichroism of native (ZnZn-) and apophospholipase C (Bacillus cereus) indicated marked protein unfolding at denaturant concentrations of 1.4–1.8 M and 0.1–0.6 M, respectively. With the apoenzyme near u.V. region circular dichroism bands remained even after all ordered structure appeared to have been lost. Apophospholipase C bound two equivalents of Ni²⁺, Cd²⁺, Co²⁺, Mn², Pb²⁺ or Cu²⁻, with only the latter metal causing marked changes either in circular dichroism or protein fluorescence relative to the native enzyme. Stability in guanidinium chloride for the metalloforms of phospholipase C decreased in the order: ZnZn->ZnCo->NiNi->CoCo->PbPb->CdCd->MnMn-apoenzyme.     

Biochemical and biophysical studies on cytochrome aa3 VIII. Effect of cyanide on the catalytic activity

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa₃ in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10³ M⁻¹·s⁻¹ and a K_i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa₃−O₂ system a biphasic reduction of cytochrome c occurs corresponding to an initial K_i of 0.8 μM and a final K_i of about 0.1 μM for the cytochrome aa₃−cyanide reaction.

3. 3. The inhibited species (a²+a₃³⁺HCN) is formed when a²⁺a₃³⁺ reacts with HCN, when a²⁺a₃²⁺HCN reacts with oxygen, or when a³⁺a₃³⁺HCN (cyano-cytochrome aa₃) is reduced. Cyanide dissociates from a²⁺a₃³⁺HCN at a rate of 2·10⁻³ s⁻¹ at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a²⁺a₃³⁺ than to a³⁺a₃³⁺.

Effect of the homokaryotic or heterokaryotic state of the uvs-2 allele in Neurospora crassa on mitomycin C-induced killing and ad-3 mutation

Mitomycin C (MC) was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 4 dikaryons of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on MC-induced killing and ad-3 mutation. These dikaryons were homokaryotic for uvs-2⁺ (H-12), homokaryotic for uvs-2 (H-59), and heterokaryotic for uvs-2/uvs-2⁺ (H-70 and H-71). MC induced killing and ad-3 mutation in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a great increase in the killing and mutagenic activities of MC. This increased sensitivity to MC-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is a different effect than that noted by others for a defect in nucleotide excision-repair in Escherichia coli and Salmonella typhimurium or in human cells. The dikaryons heterokaryotic for uvs-2/uvs-2⁺ had the same sensitivity to MC as H-12, indicating that for MC-induced killing and ad-3 mutation uvs-2 is recessive to uvs-2⁺.     

Essential role of membrane ATPase or coupling factor for anaerobic growth and anaerobic active transport in Escherichia coli

Mutants of Escherichia coli defective in coupling electron transport to synthesis of ATP (unc⁻) were isolated and screened for Mg²⁺-ATPase activity using a rapid and sensitive Millipore filtration assay. An episome (F′16) carrying ATPase genes was used to map the unc⁻ mutations near the ilv (isoleucine-valine) operon. Mutants missing membrane ATPase activity do not multiply anaerobically on glucose as energy source unless supplied with exogenous electron acceptors such as NO₃⁻. Likewise, in the absence of exogenous electron acceptors anaerobic active transport of proline is blocked. These observations suggest that membrane ATPase has an essential role in membrane functions linked to glycolysis and thus may play an important role in energy conversion in the anaerobic membrane.     

Chromosomal instability, reproductive cell death and apoptosis induced by O-methylguanine in Mex, Mex and methylation-tolerant mismatch repair compromised cells: facts and models

O⁶-Methylguanine (O⁶-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O⁶-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O⁶-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex⁻), expressing MGMT (Mex⁺) or exhibiting the tolerance phenotype (Mex⁻, methylation resistant) were compared as to their clastogenic response. Mex⁻ cells were more sensitive than Mex⁺ cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex⁺ as compared to Mex⁻ cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex⁻ cells whereas it dropped to nearly control level in Mex⁺ cells. From this we conclude that, in the first post-treatment replication cycle of Mex⁻ cells, only a minor part of aberrations (<40%) was due to O⁶-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (>90%) was caused by the lesion. Thus, O⁶-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex⁻ cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O⁶-MeG. In methylation tolerant cells, which are characterized by impaired G–T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O⁶-MeG–T mispairs is decisively involved in O⁶-MeG born chromosomal instability and recombination. We also show that Mex⁺ and methylation tolerant cells are more resistant than Mex⁻ cells with regard to induction of apoptosis, indicating O⁶-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.     

Two modules from the hypersuppressive rho mitochondrial DNA are required for plasmid replication in yeast

In the yeast hypersuppressive (HS) rho⁻ mutants most of the mitochondrial genome is deleted, but the remainder containing one of the three rep sequences is amplified. One of these sequences, rep2, and its flanking regions have been previously cloned and reported to promote autonomous plasmid replication in yeast. The present study suggests that the Ars activity associated with this HS rho⁻ mitochondrial DNA (mtDNA) fragment is due to the presence in cis of at least two modules: (i) the 11-bp consensus sequence 5′-^A_TAAA^C_TATAAA^A_T-3′, common to several ars sequences, and (ii) a palindromic sequence of the mitochondrial replicator. Proper spacing between the two modules, which varies from about 100 to 200 bp, is required for the Ars ⁺ activity.