No abstract

We examined the in vivo expression of up to 16 genes encoding for components of both glutaredoxin and thioredoxin systems and for members of the OxyR and SoxRS regulons. We demonstrated that grxA (Grx1) transcription is triggered in bacteria lacking Trx1 (trxA) and GSH (gshA) in an OxyR-dependent manner. We also indicated that, unlike OxyR, SoxR is not constitutively activated in the oxidizing environment of trxA gshA mutants. We discovered that the lack of Trx1 plus GSH increases the steady-state levels of Trx reductase (trxB) and Trx2 (trxC) transcripts. This increase and the trxB and trxC up-regulation caused by the constitutive oxyR2 allele indicate that OxyR also plays a role in the regulation of the thioredoxin pathway. On the contrary, no change in the expression of genes for Trx1, Grx2, and Grx3 was observed. Transcription of nrdAB (RRase) was not induced by oxidative stress yet was induced by hydroxyurea (RRase inhibitor). Induction level was as the enhanced nrdAB basal expression of trxA grxA mutants, indicating that RRase operation without Trx1 and Grx1 must lead to disturbances sensed as those caused by hydroxyurea. We also demonstrated an inverse relation between nrdAB expression and that of genes coding for components of both glutaredoxin (grxA, gorA) and thioredoxin (trxB, trxC) systems.

This work provides the first absolute expression patterns of genes coding for all known components of both thioredoxin (Trx) and glutaredoxin (Grx) systems in mouse: Trx1, Trx2, Grx1, Grx2, TrxR1, TrxR2, thioredoxin/glutathione reductase, and glutathione reductase. We devised a novel assay that, combining the advantages of multiplex and real-time PCR, streamlines the quantitation of the actual mRNA copy numbers in whole-animal experiments. Quantitations reported establish differences among adult organs and embryonic stages, compare mRNA decay rates, explore the significance of alternative mRNA isoforms derived from TrxR1 and Grx2 genes, and examine the time-course expression upon superoxide stress promoted by paraquat. Collectively, these quantitations show: i) unique expression profiles for each transcript and mouse organ examined, yet with some general trends like the higher amounts of mRNA species coding for thioredoxins than those coding for the reductases that control their redox states and activities; ii) continuous expression during embryogenesis with outstanding up-regulations of Trx1 and TrxR1 mRNAs in specific temporal sequences; iii) drastic differences in mRNA stability, liver decay rates range from 2.8 h (thioredoxin/glutathione reductase) to > 35 h (Trx1 and Trx2), and directly correlate with mRNA steadystate values; iv) testis-specific differences in the amounts (relative to total isoforms) of transcripts yielding the mitochondrial Grx2a and 67-kDa TrxR1 variants; and v) coordinated up-regulation of TrxR1 and glutathione reductase mRNAs in response to superoxide stress in an organ-specific manner. Further insights into in vivo roles of these redox systems should be gained from more focused studies of the mechanisms underlying the vast differences reported here at the transcript level.

We evaluated the suitability of high-throughput proteomic methods to monitor terrestrial ecosystems. Free-living Mus spretus from three sites along the "Domingo Rubio" (DR) stream were compared with mice from Doñana Biological Reserve ("Santa Olalla" lagoon (SOL) negative control), using specimens from an industrial settlement (phosphogypsum stacks (PS)) and rice fields ("Matochal" rice fields (ARZ)) as positive controls. Our 2-DE analysis showed 36 spots with significantly altered expression. Sixteen were identified by MALDI-TOF-PMF and peptide matching with Mus musculus databases. Identified proteins play different roles: cytoskeletal dynamics, proteolysis, biotransformation, oxidative-stress adaptation, and metabolism. Animals from different polluted environments showed contrasting differences in their proteomes, with specific increases and decreases in selected groups of proteins that seem to be co-ordinately regulated. Proteomic data were consistent with metal biomonitoring and conventional biomarker responses, indicating that DR (and PS/ARZ) animals sustained a heavier pollutant burden than SOL specimens and suffered a chronic oxidative stress. Whereas some protein expression differences may protect mice from pollutant toxicity, others should make them more susceptible. Transcript expression signatures agree with the documented lack of correlation between mRNA and protein levels. Nonetheless, a positive significant correlation was found between the gpx1 mRNA molecules and the intensity of one of the two identified GPX1 isospots.

No abstract

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB and nrdHIEF operons. While NrdAB is active during aerobiosis, NrdEF is considered a cryptic enzyme with no obvious function. Here, we present evidence that nrdHIEF expression might be important under certain circumstances. Basal transcript levels were dramatically enhanced (25-75-fold), depending on the growth-phase and the growthmedium composition. Likewise, a large increase of >100-fold in nrdHIEF mRNA was observed in bacteria lacking Trx1 and Grx1, the two main NrdAB reductants. Moreover, nrdHIEF expression was triggered in response to oxidative stress, particularly in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase activities (69.7-fold) and in cells treated with oxidants (up to 23.4-fold over the enhanced transcript level possessed by cells grown on minimal medium). The mechanism(s) that triggers nrdHIEF expression remains unknown, but our findings exclude putative global regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have learned about nrdHIEF expression indicates strong differences between its regulation and that of the nrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. coli might optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.

Most studies using real-time PCR are taken semiquantitatively and assume a steady level of expression forthe so-called housekeeping genes. By absolute real-time PCR we demonstrate that the transcript amounts of two of the most popular internall controls (coding GAPDH and beta-actin) fluctuate dramatically across diverse mouse or human tissues. This raises the question about the inaccuracy of these genes a squantitative references in tissue-specific mRNA profiling. Target genes chosen for absolute real-time PCR analysis are involved in DNA repair, regulation of gene expression, and oxidative stress response. Hence, they code for 8-oxoG-DNA glycosylase/AP-lyase, major AP-endonuclease, and heme oxygenase-1. Quantitations reported: i) determine mouse-to-mouse variability in basal gene expression, ii) establish organ- and embryo-associated differences in mouse, iii) compare mouse and human tissue-specific profiles, iv) examine the time course (30-240 min) expression in liver and lung of mice treated with paraquat (superoxide generator) at 30 mg kg(-1) (one half LD50 value), and v) explore the utility of absolute real-time PCR in field studies with genetically diverse mice. We conclusively establish that real-time PCR is a highly sensitive and reproducible technique for absolute quantitation of transcript levels in vivo and propose its use to quantitate gene expression modulation under mild physiological exposures and for field epidemiological studies.

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.