Biomedical subjects

C Pueyo

Publications and source records attributed to C Pueyo.

At least 37 records · Page 2Linked to original sources

PubMed1997

The influence of DNA repair by Ogt alkyltransferase on the distribution of alkylnitrosourea-induced mutations in Escherichia coli.

To determine the influence of DNA repair by Ogt alkyltransferase on the distribution of alkylnitrosourea-induced mutations, we have analysed in Ogt-proficient and Ogt-deficient bacterial strains the DNA sequence changes of a total of 357 independent mutations occurring within the initial part of the lacl gene of Escherichia coli. The majority (>80%) of mutations induced by either N-ethyl-N nitrosourea (ENU) or N-methyl-N-nitrosourea (MNU) in the two genetic backgrounds were G:C --> A:T transitions, consistent with the predominant role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. The analysis of the distribution of G:C --> A:T transitions induced by ENU in Ogt+ and Ogt bacteria reveals an influence of the 5'-flanking base at the level of repair by Ogt alkyltransferase. The Ogt protein appears more efficient at repairing O6-ethylguanine lesions, which are flanked 5' by a G or C, in agreement with previously reported data from our group for ethylmethane sulfonate. In contrast, no preference could be inferred for the repair of O6-methylguanine lesions by Ogt protein. These results seem to indicate that the preference of the Ogt alkyltransferase to repair certain DNA sequences might be a function of the size of the alkyl group. The importance of the alkyl group length has been described also at the level of the (A)BC excinuclease machinery that seems to have a DNA sequence specificity opposite to that of Ogt alkyltransferose.

Base Sequence↗

PubMed1997

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene.

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the supF gene of Escherichia coli were recovered from bacteria deficient in nucleotide excision repair and in DNA-alkyltransferase activity. Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7-fold above the spontaneous value. A total of 73 independent supF- mutants were sequenced. The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle-vector approach (in vitro supF mutagenesis). In vivo CCNU mutagenesis in E. coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C-->A:T transitions. A substantial proportion (6/18) of CCNU-induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class. Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C-->A:T transitions (39/53). This mutational change was attributed to the mispair potential of the O6-chloroethylguanine lesion with thymine. Most G:C-->A:T transitions (34/39) were located at three 5'-GG-3' hotspot sites (positions 123, 160, and 168). The distribution of hotspot sites for G:C-->A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event. Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6-chloroethylguanine formation, reducing its incidence in the transcribed DNA strand.

Base Composition↗

PubMed1997

Spontaneous ascent of the testis.

OBJECTIVE: To report confirmed cases of spontaneous testicular ascent in patients with a cryptorchid testis which had been previously placed in the scrotum. PATIENTS AND METHODS: Between 1972 and 1992, 46 cryptorchid testes (36 patients), which were previously verified to be in the scrotum by staff paediatricians or paediatric surgeons, were treated surgically. The clinical, surgical and anatomicopathological records were reviewed to determine testicular position and associated conditions, and biopsies were taken from 35 testes. RESULTS: The mean time between the last "normal' exploration until the testes ascended was 4.5 years. Before ascent, the testes were confirmed to be in the scrotum by up to seven separate recorded examinations. During surgical treatment, an open processus vaginalis was found in 18 testes. The mean (SD) tubular fertility index was 40 (37)%. CONCLUSIONS: This study confirms that a previously descended testis can ascend spontaneously. The clinical and anatomicopathological characteristics of these testes were similar to those of the cryptorchid testis.

Child↗

PubMed1997

Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes.

Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6-alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector-transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane-derived species to alkylate Trx.

Animals↗

PubMed1997

Mutation spectra analysis suggests that N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-induced lesions are subject to transcription-coupled repair in Escherichia coli.

To determine the influence of some bacterial DNA repair pathways on the mutagenic and the lethal effects of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), pZ189 plasmids treated in vitro with 2 mM CCNU were transfected into Escherichia coli strains with different repair capacities (uvr+ada+ogt+, uvr-ada+ogt+, and uvr-ada-ogt-). Despite the differences in repair capacities, no statistically significant difference in survival and mutability was observed among the tested strains. One hundred and sixty-six CCNU-induced supF mutants were isolated and sequenced. All mutants were characterized by single base-pair substitutions, most of which (more than 96%) were GC-->AT transitions (the mutated G being almost exclusively preceded 5' by a purine). Mutation distribution was not random. Position 160 (5'-GGT-3', nontranscribed (NT) strand) was a uvr+ada+ogt(+)-specific hot-spot. Position 123 (5'-GGG-3', NT strand) was a common hot-spot but significantly more mutable in repair-proficient strains than in repair-deficient strains. Conversely, position 168 (5'-GGA-3', transcribed (T) strand) was significantly more mutable in repair-deficient strains than in repair-proficient strains. By applying a computer program for comparison of mutational spectra, we found that the uvr+ mutational spectrum was significantly different from those obtained in uvr- strains, whereas in the uvr- background, no difference was observed between mutation spectra in ada+ogt+ versus ada-ogt- strains. Our results are consistent with the hypothesis that O6-alkylguanine is responsible for most mutations observed in all strains. The results also indicate that excision repair modulates the distribution of GC-->AT transitions. The fact that mutations at G lesions on the T strand were significantly less frequent in uvr+ than in uvr- strains suggests that CCNU-induced premutational lesions are susceptible to strand-preferential repair in E. coli.

Base Sequence↗

PubMed1996

The levels of ribonucleotide reductase, thioredoxin, glutaredoxin 1, and GSH are balanced in Escherichia coli K12.

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.

Bacterial Proteins↗

PubMed1996

A mimetic of the RGDF-peptide [arginine-glycine-aspartic acid-phenylalanine] blocks aggregation and flow-induced platelet deposition on severely injured stenotic arterial wall. Effects on different animal models and in humans.

8-Guanidino-octanoyl-aspartic acid-phenylalanine (SC-49992), a mimetic of the tetrapeptide arginine-glycine-aspartic acid-phelylalanine, inhibits fibrinogen and vitronectin binding to GP IIb/IIIa. SC-49992 effects on impedance and optical aggregation were compared in different species (human, porcine and dog), SC-49992 induced significant inhibition both in whole blood and PRP aggregation, in all species; however, porcine platelets had a SC-49992 IC50 = 2.5 mM while human and dog platelets had a significant lower IC50 (1 microM and 1.5 microM, respectively). Inhibition of flow-mediated platelet deposition on severely injured vessels was studied in porcine blood at high and low shear rates for 5 minutes. Additionally, studies were performed in vessels with 80% stenosis. SC-49992 reduced platelet deposition both at high and low shear rate in parallel streamlined flow and in stenotic conditions. The lower affinity of porcine platelets for SC-49992 seems to be due to a species difference at the GPIIb/IIIa receptor RGD-sequence level.

Amino Acid Sequence↗

PubMed1996

Contribution of ogt-encoded alkyltransferase to resistance to chloroethylnitrosoureas in nucleotide excision repair-deficient Escherichia coli.

We investigated the relative contribution of the two Escherichia coli DNA alkyltransferases (ATases) to the increased sensitivity of ATase-deficient bacteria to the mutagenic and lethal effects of chloroethylnitrosoureas (CNU). The ogtencoded protein was the principal determinant in resistance to the mutagenic effects of CNU in E.coli. Thus, only when the ogt gene was inactivated was sensitivity to mutagenesis greatly increased; the contribution of inactivation of the ada gene was relatively minor. Furthermore, induction of the adaptive response provided essentially no protection against CNU mutagenesis in either an ogt+ or ogt- background. Finally, overexpression of the ogt gene into ogt- ada- double mutants provided the greatest protection against CNU; introduction of the full-length or truncated ada gene was protective, but to a much lesser extent. Mammalian ATases were not as protective against mutation induction by CNU as Ogt, even though they were apparently expressed at higher level. In order of effectiveness the ATases ranked Ogt > human > truncated Ada = Ada > rat. This order was not observed in the protection against killing by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, where truncated Ada = human > Ogt > rat = Ada. Higher mutation frequency and toxicity were observed in uvr- mutants, suggesting that one or more of the potentially mutagenic and/or toxic lesions are also substrates for the excision repair proteins.

Bacterial Proteins↗

PubMed1996

Mutational specificity of aflatoxin B1. Comparison of in vivo host-mediated assay with in vitro S9 metabolic activation.

An intrasanguineous host-mediated assay was used to determine the pattern of mutagenesis induced by the carcinogen aflatoxin B1 in the lacI gene of Escherichia coli recovered from rat liver. To investigate the influence of different types of metabolic activation, the mutation spectrum induced by AFB1 activated in vitro by a commercially prepared S9 microsomal fraction from Aroclor 1254-treated rats was also obtained. A total of 281 forward mutations affecting the N-terminal region of the lacI gene were characterized by DNA sequencing analysis. AFB1 induced similar type of mutations with similar site specificity when activated by the standard S9 fraction or by employing a rat host-mediated assay. These results indicate the ability of the in vitro S9 fraction to mimic the in vivo metabolism, suggesting that the same active metabolite, presumably AFB1 8,9-epoxide, is responsible for generating a similar pattern of DNA damage, as reflected in the similarity of mutational spectra. For both activation systems, most mutations (>90%) were base substitutions that occurred primarily at G:C pairs. Somewhat over one-half of G:C targeted substitutions were GC>TA transversions, other mutations being evenly divided between G:C>AT transitions and GC>CG transversions. The mutational specificity exhibited by activated AFB1 can be explained by incorporation of different bases opposite a single type of non-instructive lesion during error-prone DNA synthesis. To what extent the mutations are due to the main adduct (AFB1-N7-Gua), its imidazole-ring-opened derivative or an apurinic site remains unknown.

Aflatoxin B1↗

PubMed1995

Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants.

Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism. To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used. Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats. The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system. This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics. The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic. The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity. The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.

2-Acetylaminofluorene↗

PubMed1995

Role of classical nitroreductase and O-acetyltransferase on the mutagenicity of nifurtimox and eight derivatives in Salmonella typhimurium.

This study investigates the mutagenicity of nifurtimox (NFX) and eight analogues in Salmonella typhimurium indicator strains that possess different levels of classical nitroreductase or O-acetyltransferase activities. The NFX analogues tested replace the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1G); pyrazol-1-yl (1B); benzimidazol-1-yl (1E); 1,2,4-triazol-4-yl (1D); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (1I); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (1H); 1-adamantyl (ADA); and 4,6-diphenylpyridin-1-yl-2-one (1K). In the genetic backgrounds of the standard Ames tester strains TA98 and TA100, these bacteria combine the L-arabinose resistance forward mutation assay (Ara test) with a deficiency or overproduction of either nitroreduction or O-acetylation. The Ara test revealed, in agreement with previous findings, important differences between TA98 and TA100 and demonstrated, moreover, that these genetic differences are of significance in mutagenicity testing with nitrofuran compounds. The Ara test also indicated dissimilarities between the metabolic activation of NFX and its analogues, these compounds being classified in three different groups according to their mutagenicity toward strain BA14 (genetic background of TA98) and its derivatives. The first group included analogues (1G, 1E, 1I, and ADA) that showed similar mutagenic potency in all bacterial strains. These compounds are considered not to be substrates for both classical nitroreductase and O-acetyltransferase. The second group included compounds (analogues 1B and 1K, and the reference drug NFX) with increased mutagenicity toward the strain overproducing the classical nitroreductase, and/or reduced mutagenicity toward the corresponding deficient bacteria. These compounds are considered to be activated by the classical nitroreductase. The third group (analogues 1D and 1H) was activated by bacterial O-acetyltransferase, and consequently showed increased and decreased mutagenicity with the particular overproducer or deficient bacterial strain, as compared to their isogenic parentals. Previous reports have pointed out interest in NFX analogue 1H as a promising candidate for the replacement of NFX. The present study further enhances the putative interest of compound 1H, based on the different metabolic activation pathway exhibited by this analogue as compared to the parental drug, NFX.

Acetyltransferases↗

PubMed1995

ogt alkyltransferase enhances dibromoalkane mutagenicity in excision repair-deficient Escherichia coli K-12.

We examined the role of the O6-alkylguanine-DNA alkyltransferase encoded by ogt gene in the sensitivity of Escherichia coli to the mutagenic effects of the dibromoalkanes, dibromoethane and dibromomethane, by comparing responses in ogt- bacteria to those in their isogenic ogt+ parental counterparts. The effects of the uvrABC excision-repair system, the adaptive response, mucAB and umuDC mutagenic processing, and glutathione bioactivation on the differential responses of ogt- and ogt+ bacteria were also studied. Mutation induction was monitored by measuring the frequency of forward mutations to L-arabinose resistance. Induced mutations occurred only in excision repair-defective strains and were totally (with dibromomethane) or substantially (with dibromoethane) dependent on the alkyltransferase (ATase) encoded by the ogt gene. An increased mutagenic response to both dibromoalkanes was also seen in ogt- bacteria that overexpressed the ogt protein from a multicopy plasmid, indicating that the differences in mutability between ogt+ and ogt- bacteria were not dependent on the ogt- null allele carried by the defective strain. The ATase encoded by the constitutive ogt gene was more effective in promoting dibromoalkane mutagenicity than the ada ATase induced by exposure to low doses of a methylating agent. The mutagenicity promoted by the ogt ATase was dependent on both glutathione bioactivation and SOS mutagenic processing. To our knowledge, this paper presents for the first time evidence that DNA ATases, in particular the ATase encoded by the ogt gene, can increase the mutagenic effects of a DNA-damaging agent. The mechanism of this effect has yet to be established.

DNA Repair↗

PubMed1995

Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in the Escherichia coli lacl gene of O6-alkylguanine-DNA alkyltransferase-proficient and -deficient strains.

Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the lacl gene of Escherichia coli were recovered from bacteria proficient (Ogt+ Ada+) and deficient (Ogt- Ada-) in O6-alkylguanine-DNA alkyltransferase activity. A CCNU dose of 1 mM was selected for DNA sequence analysis. A total of 245 induced mutations were characterized. The mutations were almost exclusively (95%) GC-->AT transitions, indicating that CCNU-induced mutations arose in bacteria primarily from misreplication of O6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents. The distribution of CCNU-induced GC-->AT mutations was significantly altered by the presence of DNA alkyltransferase activity (P = 0.01). In the Ogt+ Ada+ mutational spectrum, guanines flanked on both sides by A:T base-pairs were on average 2.8 times more likely to mutate than those flanked by G:C base-pairs on at least one side. This bias disappeared in the Ogt- Ada- genetic background, thereby providing evidence that O6-chloroethylated guanines adjacent to G:C base-pairs are better targets for bacterial alkyltransferase than those not adjacent to G:C base-pairs. We recently reported a similar bias for ethyl methanesulfonate, strengthening the idea that CCNU is acting as a simple ethylating compound. In summary, this paper presents for the first time evidence that DNA repair by O6-alkylguanine-DNA alkyltransferases plays a major role in removing lesions responsible for GC-->AT transitions induced by CCNU, influencing their ultimate distribution with respect to sequence context.

Antineoplastic Agents↗

PubMed1995

Mathematical parameters for quantification of mutational responses in bacteria.

This paper introduces a new parameter, derivable from dose-response data for induced mutagenesis in bacteria, that can be used to quantify mutational responses in short-term tests. We called this parameter the mutational response of the bipartite experimental system (agent plus cells). We defined it as being jointly proportional to the efficiency of the mutagen and the sensitivity of the test. We show how this quantity can be used to rank order chemical carcinogens on the basis of their mutagenicity and to determine the strength of any quantitative correlation that may exist between mutagenicity in bacteria and carcinogenicity in rodents. We find that this particular measure of mutational response for 10 direct-acting monofunctional alkylating agents correlates remarkably well with the rodent carcinogenicity of these chemicals measured in terms of their reciprocal TD50 values.

Alkylating Agents↗

PubMed1995

DNA repair by Ogt alkyltransferase influences EMS mutational specificity.

Forward mutations induced by ethylmethane sulfonate (EMS) in the lacI gene of Escherichia coli were recovered from bacteria proficient or deficient in the alkyltransferase encoded by the constitutive ogt gene. EMS doses of 100 or 200 mM (Ogt+) and of 50 mM (Ogt-) were selected from the corresponding dose-response curves for DNA sequence analysis. A total of 239 induced mutations affecting the N-terminal region of the lacI gene were characterized. All mutations were G:C-->A:T transitions, consistent with the predominant role of the O6-ethylguanine miscoding lesion in mutagenesis by EMS. In the Ogt+ spectrum at the lowest tested dose of 100 mM EMS, guanines preceded by an A or T base at the 5' side were on average 3.2 times more likely to mutate than those preceded by a G or C base. This bias diminished at the higher EMS dose (200 mM) and disappeared in the Ogt- genetic background. Previously reported data for Ogt+ bacteria in a Uvr-proficient background show an opposite bias in favor of mutations at guanines preceded by a G or C base. The overall 5' flanking base influence was estimated as 8-fold. These data suggest that DNA repair by Ogt alkyltransferase plays an important role in the processing of ethylation-induced lesions responsible for GC-->AT transitions, influencing their ultimate distribution with respect to sequence context. The data further suggest that Ogt and UvrABC excision repair, the two major mechanisms of protection against the biological consequences of long-chain alkylating agents, show different DNA sequence specificity and that the relative importance of these two systems is highly dependent upon the chemical dose.

Bacterial Proteins↗

PubMed1994

Two additional glutaredoxins exist in Escherichia coli: glutaredoxin 3 is a hydrogen donor for ribonucleotide reductase in a thioredoxin/glutaredoxin 1 double mutant.

Thioredoxin (Trx) and glutaredoxin (Grx1) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. The viability of a double mutant lacking both Trx and Grx1 implies the presence of a third, unknown hydrogen donor. This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grx1 (delta trxA, grx::kan). Affinity chromatography was used to bind glutaredoxin on a glutathione-containing thiol-Sepharose column. The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000. Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys. The proteins are therefore referred to as Grx2 and Grx3. The low hydrogen donor activity for ribonucleotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive. As a hydrogen donor for E. coli ribonucleotide reductase, Grx3 showed approximately the same Km value (0.35 microM) as Grx1, whereas its Vmax value was only 5% that of Grx1. The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a delta trxA, grx double mutant provides an explanation for its viability and deoxyribonucleotide biosynthesis. The physiological functions of Grx2 and Grx3 remain to be determined.

Amino Acid Sequence↗

PubMed1994

Null thioredoxin and glutaredoxin Escherichia coli K-12 mutants have no enhanced sensitivity to mutagens due to a new GSH-dependent hydrogen donor and high increases in ribonucleotide reductase activity.

This work investigates whether a mutator phenotype is associated to the simultaneous deficiency in thioredoxin and glutaredoxin, the two known hydrogen donors of ribonucleotide reductase. To this end, new Escherichia coli K-12 strains carrying delta trxA and/or grx::kan null mutations were constructed to monitor mutagenesis by selecting forward mutations to L-arabinose resistance. Highly sensitive and specific enzyme-linked immunoassays were developed to confirm that trx-grx- cells lacked thioredoxin and glutaredoxin. A number of remarkable properties were observed in the newly constructed thioredoxin- and glutaredoxin-deficient bacteria compared with the wild type cells. Thus, they (i) grew on minimal medium plates, suggesting that the presence of thioredoxin and glutaredoxin may not be absolutely essential for sulfate reduction; (ii) showed normal mutagenic sensitivities toward a wide variety of DNA-damaging agents, as compared with wild type cells and trx- or grx- single mutants; (iii) displayed 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous ribonucleotide reductase, respectively; and (iv) showed very high levels of ribonucleotide reductase activity, which was increased from 19- to 23-fold. The existence of a new glutathione-dependent hydrogen donor for ribonucleotide reductase and the high activity levels of this enzyme in trx-grx- defective cells could explain that thioredoxin and the first discovered glutaredoxin are not essential for deoxyribonucleotide synthesis, even under mutagenic stress.

Bacterial Proteins↗

PubMed1994

Mutagenicity testing in Salmonella typhimurium strains possessing both the His reversion and Ara forward mutation systems and different levels of classical nitroreductase or O-acetyltransferase activities.

The induction of forward mutations to L-arabinose resistance (AraR) and of reversions to histidine prototrophy (His+) can be quantitatively compared in Salmonella typhimurium BA strains. The BA bacteria carry the araD531 allele required for the Ara assay and a his auxotrophy (hisD3052 or hisG46) required for the His assay. In this study, 2 new sets of BA indicator strains have been constructed in order to combine the Ara forward and the His reverse mutation assays of S. typhimurium with deficiency, or over-production, in either classical nitroreductase or O-acetyltransferase for mutagenicity testing of nitro-containing chemicals. Nine mutagens with different chemical structures were tested to compare the specific mutagenic sensitivities of the new constructions with those of the parental and of the conventional TA indicator bacteria. The Ara test, which responded with high sensitivity to all chemicals tested, revealed important differences between the standard tester strains TA98 and TA100 with respect to the activation of mutagens considered to be dependent on classical nitroreductase activity. Total correspondence was found between the specific mutagenic sensitivities of the defective and the overproducing bacteria in the genetic background of TA98 but not in that of TA100. In the genetic background of TA100, chemicals such as nitrofurantoin and nitrofurazone displayed 10-fold reduced mutagenicity to the "classical nitroreductase" defective strain without increasing mutagenicity to the corresponding overproducing bacteria. This discrepancy might be attributed to the greater nitroreduction capability of strain TA100 (68.12 nmole/min/mg protein) as compared to TA98 (24.42 nmole/min/mg protein), by assuming that nitrofurantoin and nitrofurazone are such good substrates for classical nitroreductase that the additional enzyme activity produced from the corresponding overexpressing plasmid when present in TA100 no longer affected their metabolic activation. We propose that the Ara forward mutation test carried out in the set of over-producing bacteria constructed in the genetic background of TA98 might play a role for routine testing of large number of samples. The isogenic defective strains could be used in cases of uncertain results with the corresponding over-producing bacteria. Finally, the reversion of the his alleles accompanying the Ara assay in the BA strains could play a role in assessing the presence of mixtures of chemicals with different mutagenic specificity in samples of environmental relevance such as urban air, foods, and water.

Arabinose↗