Joaquín Abián

COMPLEXES OF IRON WITH PHENOLIC COMPOUNDS FROM SOYBEAN NODULES AND OTHER LEGUME TISSUES : PROOXIDANT AND ANTIOXIDANT PROPERTIES

The low-molecular-mass fraction of the soybean nodule cytosol contains Fe capable of catalyzing free radical production through Fenton chemistry. A large portion of the pool of catalytic Fe, measured as bleomycin-detectable Fe, was characterized as complexes of Fe with phenolic compounds of three classes: phenolic acids, cinnamic acids, and flavonoids. Many of these compounds, along with other phenolics present in legume tissues, were used for a systematic structure-activity relationship study. All phenolics tested were able to chelate Fe, as judged from their inhibitory effect on site-specific deoxyribose degradation (minus EDTA assay). However, only those having catechol, pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe3+ at pH 5.5. The same phenolics promoted oxidative damage to DNA (bleomycin assay) and to deoxyribose (plus EDTA assay), but inhibited linolenic acid peroxidation by chelating and reducing Fe3+ and by neutralizing lipid radicals. Also, phenolics having a pyrogallol nucleus attenuated the free radical-mediated inactivation of glutamine synthetase, which was used as a model system, by chelating Fe2+. It is reasoned that under the microaerobic (10-20 nM O2) and acidic (pH 5.5-6.4) conditions prevailing in nodules, phenolics are likely to act primarily as antioxidants, decreasing oxidative damage to biomolecules.

Comparison of conventional, narrow-bore and capillary liquid chromatography/mass spectrometry for electrospray ionization mass spectrometry : Practical considerations

Electrospray ionization (ESI) is nowadays the most important technique for on-line liquid chromatography/mass spectrometry (LC/MS) coupling. Different ESI probe designs including the microESI and pneumatically assisted ESI (ionspray) interfaces allow the introduction of sample at flow-rates ranging from a few hundred nl min−1 to 1–2 ml min−1. In this paper, an overview of the different LC/ESIMS devices is presented from the point of view of their flow compatibility. Several parameters for LC/ESIMS miniaturization and its effects on sensitivity are considered from the practical point of view. Low-flow ESI requires the use of narrow-bore and capillary columns. Sample preconcentration microdevices are recommended in order to circumvent some miniaturization drawbacks such as the low optimum injection volume and the low column capacity. Some considerations on fused-silica microcolumn and microESI needle construction are also presented. Copyright

Roles of hnRNP A1, SR proteins, and p68 helicase in c-H-ras alternative splicing regulation.

ABSTRACT Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1s inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.

Fish proteome analysis: model organisms and non-sequenced species.

In the last decade, proteomic technologies have been increasingly used in fish biology research. Proteomics has been applied primarily to investigate the physiology, development biology and the impact of contaminants in fish model organisms, such as zebrafish (Danio rerio), as well as in some commercial species produced in aquaculture, mainly salmonids and cyprinids. However, the lack of previous genetic information on most fish species has been a major drawback for a more general application of the different proteomic technologies currently available. Also, many teleosts of interest in biological research and with potential application in aquaculture hold unique physiological characteristics that cannot be directly addressed from the study of small laboratory fish models. This review describes proteomic approaches that have been used to investigate diverse biological questions in model and non‐model fish species. We will also evaluate the current possibilities to integrate fish proteomics with other “omic” approaches, as well as with additional complementary techniques, in order to address the future challenges in fish biology research.

The coupling of gas and liquid chromatography with mass spectrometry

The coupling of gas and liquid chromatography with mass spectrometry is addressed from the historical point of view. This paper discusses the more important coupling interfaces developed for GC/MS and LC/MS, including the moving belt interface, direct liquid introduction interfaces, thermospray and atmospheric pressure ionization interfaces such as atmospheric pressure chemical ionization and electrospray. Copyright

Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues

During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro‐oxidant hydrogen peroxide (H2O2) or the glutathione‐depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non‐reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy‐terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.

Quantitative peptide bioanalysis using column-switching nano liquid chromatography/mass spectrometry

Many endogenous peptides are circulating in bodily fluids at the low pmol l-1 range, placing high demands on the bioanalytical procedure. In order to analyze these minute concentrations in complex matrices, a miniaturized liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) bioanalysis method was developed using custom-made nanoLC columns (75 microns i.d.) and a micro-electrospray interface (micro ESI). To be able to analyze large sample volumes in order to cope with low biological analyte concentrations, the nanoLC/ESI-MS method was coupled to an on-line preconcentration (PC) system based on a strong anion-exchange material. This method was used to analyze endothelin peptides (ETs) in complex matrices, which are potent vasoconstrictors of M(r) approximately 2500 Da. The ET isoforms could be simultaneously analyzed with detection limits down to 30 pmol l-1 in cell supernatants (1.5 fmol on column). The method was linear from 50 to 2000 pmol l-1 with correlation coefficients of 0.99 for two of the three endothelin isoforms. Several other parameters, such as matrix effects and recovery, were also investigated.

Urine Proteomics to Detect Biomarkers for Chronic Allograft Dysfunction

Despite optimal immunosuppressive therapy, more than 50% of kidney transplants fail because of chronic allograft dysfunction. A noninvasive means to diagnose chronic allograft dysfunction may allow earlier interventions that could improve graft half-life. In this proof-of-concept study, we used mass spectrometry to analyze differences in the urinary polypeptide patterns of 32 patients with chronic allograft dysfunction (14 with pure interstitial fibrosis and tubular atrophy and 18 with chronic active antibody-mediated rejection) and 18 control subjects (eight stable recipients and 10 healthy control subjects). Unsupervised hierarchical clustering showed good segregation of samples in groups corresponding mainly to the four biomedical conditions. Moreover, the composition of the proteome of the pure interstitial fibrosis and tubular atrophy group differed from that of the chronic active antibody-mediated rejection group, and an independent validation set confirmed these results. The 14 protein ions that best discriminated between these two groups correctly identified 100% of the patients with pure interstitial fibrosis and tubular atrophy and 100% of the patients with chronic active antibody-mediated rejection. In summary, this study establishes a pattern for two histologic lesions associated with distinct graft outcomes and constitutes a first step to designing a specific, noninvasive diagnostic tool for chronic allograft dysfunction.

High-performance liquid chromatography—thermospray mass spectrometry of ten sulfonamide antibiotics: Analysis in milk at the ppb level

Ten sulfonamide antibiotics including sulfanilamide (SNL), sulfamethazine (SMZ), sulfamethizole (SMTZ), sulfachloropyridazine and sulfaquinoxaline (SQX), were analyzed by thermospray (TSP) mass spectrometry on-line with a high-performance liquid chromatography-UV detection system. Except for the pairs SMZ-SMTZ and sulfadimethoxine-SQX, the standards were resolved in both the UV and TSP profiles. Co-eluting compounds could be differentiated in TSP by their different relative molecular masses. The [M+H]+ ion was the base peak for all the standards except SNL, which showed an [M+NH4]+ ion. Collision-induced dissociation of the [M+H]+ ions afforded daughter ion spectra characterized by common ions at m/z 92, 108 and 156, and ions derived from the amine substituent ([MH-155]+). TSP detection limits [signal-to-noise ratio (S/N) > 3] were below 20 ng (scan mode), 2 ng (selected reaction monitoring, daughter ions from [M+H]+) and 400 pg (selected ion monitoring). UV detection limits were ca. 2 ng (S/N > 5). Results obtained from the multi-residue analysis of spiked cow milk samples at the low ng/ml level are presented.

Flavin excretion from roots of iron-deficient sugar beet (Beta vulgaris L.)

The characteristics of flavin excretion from iron-deficient sugar-beet roots have been studied. Roots from iron-deficient sugar beet excreted flavins when plants were allowed to decrease the pH of the nutrient solution, but not when plants were grown in nutrient solutions buffered at high pH. As shown by reversed-phase high-performance liquid chromatography, the two major flavins whose excretion was induced by iron deficiency were different from riboflavin, FMN and FAD. These flavins have been identified as riboflavin 3′-sulfate and riboflavin 5′-sulfate by electrospray-mass spectrometry, inductively coupled plasma emission spectroscopy, infrared spectrometry and1H-nuclear magnetic resonance. We have characterized the time courses of accumulation of the different flavins in the nutrient solution and considered several possible roles for flavin excretion in iron acquisition.