Juan Jurado

Expression Analysis of the nrdHIEF Operon from Escherichia coli: conditions that trigger the transcript level in vivo

Escherichia coli has two aerobic ribonucleotide reductases encoded by the nrdAB andnrdHIEF 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 growth-medium 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, nrdHIEFexpression 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 nrdHIEFexpression 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 thenrdAB operon and of genes coding for components of both thioredoxin/glutaredoxin pathways. We propose that E. colimight optimize the responses to different stimuli by co-evolving the expression levels for its multiple reductases and electron donors.

Proteomic profile of the skin mucus of farmed gilthead seabream (Sparus aurata)

UNLABELLED Fish skin mucus is the first line of defense against infections and it discriminates between pathogenic and commensal bacterial strains. Mucus composition varies amongst fish species and is influenced by endogenous and exogenous factors. This study describes the first proteome map of the epidermal mucus of farmed gilthead seabream (Sparus aurata). We used an integrative proteomic approach by combining a label-free procedure (LC-MS/MS) with the classical 2-DE-PMF-MS/MS methodology. The identified mucosal proteins were clustered in four groups according to their biological functions. Structural proteins (actins, keratins, tubulins, tropomyosin, cofilin-2 and filamin-A) and metabolic proteins (ribosomal proteins, proteasomal subunits, NACA, VCP, histones, NDPK, transferrin, glycolytic enzymes, ATP synthase components, beta-globin, Apo-A1 and FABP7) were the best represented functional categories. We also found proteins involved in stress response (WAP65, HSPC70, Cu,Zn-SOD, and PRDX1 and PRDX2) and signal transduction (PP2A 65kDa regulatory subunit, 14-3-3 protein beta/alpha, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, RhoGDI and PEBP1). Most of the identified proteins address different aspects of the innate immune response. Additionally, we analyzed bacterial peptides identified in the skin mucus of healthy S. aurata. These results revealed that genera belonging to the Lactobacillales order constitute the most abundant microorganism populations in this habitat. BIOLOGICAL SIGNIFICANCE This work shows that proteomic methods can be used to characterize fish skin mucus. Using a coupled approach of LC-MS/MS and a 2-DE-PMF-MS/MS, we have obtained the first comprehensive view of the skin mucosal proteome of S. aurata, a fish species that is economically relevant for Mediterranean aquaculture. We identified a panel of proteins involved in a variety of biological functions, particularly in the innate immune response. Furthermore, to our knowledge, this is the first time a proteomic approach has been used to examine the microbiota in the skin mucus of a fish species. Overall, these results support further immunological researches in S. aurata and are relevant for the culture of this important fish species.

The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease.

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

Metallomics integrated with proteomics in deciphering metal-related environmental issues.

The present work shows the possibilities of metallomics to characterize metal-linking proteins in Mus Musculus that could be used in environmental assessment. The laboratory mouse M. musculus is used as reference of gene/protein sequence databases to address methodological approaches based on changes in transcripts regulation, proteins expression and metalloproteins profiles in the environmental bioindicator Mus spretus that has been demonstrated to be genetically homologous to M. Musculus. A metallomic approach using size exclusion chromatography with inductively coupled plasma-mass spectrometry detection (SEC-ICP-MS) was applied to cytosolic extracts from different M. musculus organs: lung, liver, spleen, kidney, brain, testicle, hearth and muscle. The resulting profiles of metallobiomolecules revealed the presence of a Cu-binding fraction in the 7-10 kDa range which was not present in the other tissues, can be associated to low molecular mass metallothionein-like proteins. The application of reverse phase chromatography with ICP-MS detection to this fraction gives two peaks that have been isolated for later identification by tandem mass spectrometry. The mass balance of copper evaluated by ICP-MS analysis of the digested brain fractions isolated by SEC and RP chromatography reveals good recoveries of the separations. The application of 2-DE to both crude brain extract and SEC fraction (7-10 kDa) reveals the considerably reduction of the number of proteins confirming that a good purification has been attained by SEC. This integration of metallomics with proteomics and transcriptomics can be useful in further studies involving the free-living mouse M. spretus for assessment of environmental issues.

Integrated application of transcriptomics, proteomics, and metallomics in environmental studies*

Here we report a preliminary working scheme for the integrative application of transcriptomic, proteomic, and metallomic methodologies in environmental monitoring, by using as sentinel the wildlife species Mus spretus and as reference the gene/protein sequence databases from the key model species Mus musculus. We have demonstrated that the absolute transcript expression signatures quantified by reverse transcription (RT) and real-time polymerase chain reaction (PCR) of selected key genes (e.g., those coding for biotransformation enzymes) in M. spretus is a useful and reliable novel biomonitoring end-point. The suitability of commercial M. musculus oligonucleotide arrays for genome-wide transcriptional profiling in M. spretus has been also shown. Transcriptomic studies indicate considerable gene sequence similarities between both mouse species. Based on these similarities, we have demonstrated the applicability in free-living M. spretus of high-throughput proteomic methods, based on matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOFMS) analysis of tryptic 2D electrophoresis (2-DE) spot digest and peptide matching with M. musculus database. A metallomic approach based on size exclusion chromatography inductively coupled plasma-mass spectrometry (SEC-ICP-MS) was applied to trace metal-biomolecule profiles. A preliminary integration of these three -omics has been addressed to M. musculus/M. spretus couple, two rodent species that separated 3 million years ago. The integrated application of transcriptomic and proteomic data and the bidirectional use of metallomics and proteomics for selective isolation of metal-biomolecules are covered in the working scheme MEPROTRANS-triple-OMIC reported in this study.

Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species

BackgroundAlternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA.ResultsA set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins.ConclusionWe demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, ΔFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

Proteomics in HepG2 hepatocarcinoma cells with stably silenced expression of PRDX1

Peroxiredoxin 1 (PRDX1) is a member of the peroxiredoxin family. Aberrant expression of PRDX1 has been described in various cancers. We investigated the significance of this up-regulation in non-challenged hepatocellularcarcinoma (HCC) cells by establishing a HepG2 cell line stably expressing a Prdx1 shRNA. Prdx1 silencing reversed, at least partially, the tumoural phenotype of HepG2 cells, resulting in morphological changes, delayed cell growth, down-regulation of transcripts for AFP, osteopontin and β-catenin and decreased γ-glutamyl transpeptidase activity, and oppositely up-regulation of transcripts for E-cadherin and proapoptotic proteins (BAX, CASP3) and increased alkaline phosphatase and CASP3 activities. Proteomic profiling identified 16 spots differentially expressed in Prdx1-silenced cells. Most of the variations involved the down-regulation of proteins with pivotal roles in cell proliferation and differentiation, in agreement with the observed phenotypic changes. We also investigated the effect of Prdx1 silencing on thiol protein oxidation. Proteins prone to reversible cysteine oxidation play major physiological functions. Notably, the down-regulation and altered redox status of key enzymes of carbohydrate and amino acid metabolism suggested a disturbance of the Warburg effect and glutamine utilization, two major pathways in the proliferation of tumour cells. Overall, these observations suggest that PRDX1 acts as a pro-cancer protein in HCC HepG2 cells.

Identification of proteins containing redox-sensitive thiols after PRDX1, PRDX3 and GCLC silencing and/or glucose oxidase treatment in Hepa 1–6 cells

The oxidation and reduction of cysteine thiols are thought to be a major mechanism for redox regulation. The aim of this study was to identify proteins with reactive thiols and determine their oxidation profiles under oxidative stress induced by simultaneous silencing of antioxidant defences (peroxiredoxin-1, peroxiredoxin-3, and the catalytic subunit of the glutamate-cysteine ligase), and/or treatment with glucose oxidase (GO). Using an approach that combined the labelling of reversibly oxidised cysteines, 2-DE protein separation and MS analysis, we identified 26 proteins with cysteines prone to reversible oxidation belonging to different functional classes. Among these proteins are those that have not been previously recognised as reversible oxidation targets, including cytoplasmic aspartate aminotransferase, proteasome subunit alpha type-6, heterogeneous nuclear ribonucleoproteins isoA2/B1 and A/B, and histidine triad nucleotide-binding protein 1. We provide the first evidence of reversible oxidation for specific cysteines, including Cys112 and Cys146 in glutamate dehydrogenase 1, Cys17 in actins, Cys5 in protein disulfide-isomerase A3, and Cys267 in the heat shock cognate 71 kDa protein. Silencing induced lower oxidative stress than GO treatment. Nevertheless, we detected some proteins particularly sensitive to oxidation by silencing. We hypothesised that these proteins may play a role in regulatory mechanisms by redox stress.

Influence of DNA repair by (A)BC excinuclease and Ogt alkyltransferase on the distribution of mutations induced by n-propyl-N-nitrosourea in Escherichia coli

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increment in mutation induction by N‐propyl‐N‐nitrosourea (PNU). Irrespective of the presence or the absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0–8 mM PNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Some increased susceptibility to mutagenesis by PNU was detected in Uvr− Ogt+ bacteria, but the Uvr− Ogt− double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM PNU within the initial part of the IacI gene were recovered from Uvr+ Ogt−, Uvr− Ogt+, and Uvr− Ogt− bacteria. A total of 439 independent mutations were characterized by DNA sequence analysis. The PNU‐induced spectra were dominated by G:C→A:T transitions, consistent with the major role of the O6‐alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C→A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10−6). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5′‐flanking base and DNA‐strand associated with the PNU‐induced G:C→A:T transitions. Preferences different from those previously reported for the ethylating (ENU) and methylating (MNU) analogs were detected. We indicate that these differences might be caused by the PNU possibility of giving iso‐propyl adducts, in addition to the expected n‐propyl adducts, and by possible preferences in the initial distribution of these lesions as well as in their repair by the (A)BC excinuclease and the Ogt ATase of E. coli. Environ. Mol. Mutagen. 31:82–91, 1998.

Dietary administration of the probiotic Shewanella putrefaciens Pdp11 promotes transcriptional changes of genes involved in growth and immunity in Solea senegalensis larvae

ABSTRACT Senegalese sole (Solea senegalensis) has been proposed as a high‐potential species for aquaculture diversification in Southern Europe. It has been demonstrated that a proper feeding regimen during the first life stages influences larval growth and survival, as well as fry and juvenile quality. The bacterial strain Shewanella putrefaciens Pdp11 (SpPdp11) has shown very good probiotic properties in Senegalese sole, but information is scarce about its effect in the earliest stages of sole development. Thus, the aim of this study was to investigate the effect of SpPdp11, bioencapsulated in live diet, administered during metamorphosis (10–21 dph) or from the first exogenous feeding of Senegalese sole (2–21 dph). To evaluate the persistence of the probiotic effect, we sampled sole specimens from metamorphosis until the end of weaning (from 23 to 73 dph). This study demonstrated that probiotic administration from the first exogenous feeding produced beneficial effects on Senegalese sole larval development, given that specimens fed this diet exhibited higher and less dispersed weight, as well as increases in both total protein concentration and alkaline phosphatase activity, and in non‐specific immune response. Moreover, real‐time PCR documented changes in the expression of a set of genes involved in central metabolic functions including genes related to growth, genes coding for proteases (including several digestive enzymes), and genes implicated in the response to stress and in immunity. Overall, these results support the application of SpPdp11 in the first life stages of S. senegalensis as an effective tool with the clear potential to benefit sole aquaculture. HighlightsS. senegalensis larvae were fed the probiotic SpPdp11 during early life stages.SpPdp11 increased growth and decreased size dispersion in post‐larvae.SpPdp11 increased ALP activity and affected intestinal microbiota composition.SpPdp11 modulated expression of genes involved in growth, stress response and immunity.