Involvement of AOX and UCP pathways in the post-harvest ripening of papaya fruits.

Enhanced respiration during ripening in climacteric fruits is sometimes associated with an uncoupling between the ATP synthesis and the mitochondrial electron transport chain. While the participation of two energy-dissipating systems, one of which is mediated by the alternative oxidase (AOX) and the other mediated by the uncoupling protein (UCP), has been linked to fruit ripening, the relation between the activation of both mitochondrial uncoupling systems with the transient increase of ethylene synthesis (ethylene peak) remains unclear. To elucidate this question, ethylene emission and the two uncoupling (AOX and UCP) pathways were monitored in harvested papaya fruit during the ripening, from green to fully yellow skin. The results confirmed the typical climacteric behavior for papaya fruit: an initial increase in endogenous ethylene emission which reaches a maximum (peak) in the intermediate ripening stage, before finally declining to a basal level in ripe fruit. Respiration of intact fruit also increased and achieved higher levels at the end of ripening. On the other hand, in purified mitochondria extracted from fruit pulp the total respiration and respiratory control decrease while an increase in the participation of AOX and UCP pathways was markedly evident during papaya ripening. There was an increase in the AOX capacity during the transition from green fruit to the intermediate stage that accompanied the transient ethylene peak, while the O2 consumption triggered by UCP activation increased by 80% from the beginning to end stage of fruit ripening. Expression analyses of AOX (AOX1 and 2) and UCP (UCP1-5) genes revealed that the increases in the AOX and UCP capacities were linked to a higher expression of AOX1 and UCP (mainly UCP1) genes, respectively. In silico promoter analyses of both genes showed the presence of ethylene-responsive cis-elements in UCP1 and UCP2 genes. Overall, the data suggest a differential activation of AOX and UCP pathways in regulation related to the ethylene peak and induction of specific genes such as AOX1 and UCP1.

Inter-laboratory comparison of elemental analysis and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Part I: delta13C measurements of selected compounds for the development of an isotopic Grob-test.

This study was directed towards investigating suitable compounds to be used as stable isotope reference materials for gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) calibration. Several compounds were selected from those used in the 'Grob-test' mixture. Oxygen- and nitrogen-containing substances were added to these compounds to allow the mixture to be used as a possible multi-isotopic calibration tool for 2H/1H, 13C/12C, 15N/14N and 18O/16O ratio determinations. In this paper we present the results of delta13C measurements performed by the consortium of the five laboratories taking part in this inter-calibration exercise. All the compounds were individually assessed for homogeneity, short-term stability and long-term stability by means of EA-IRMS, as required by the bureau communitaire de reference (BCR) Guide for Production of Certified Reference Materials. The results were compared then with the GC-C-IRMS measurements using both polar and non-polar columns, and the final mixture of selected compounds underwent a further certification exercise assessing limits of accuracy and reproducibility under specified GC-C-IRMS conditions.

Analysis of 8-hydroxy-2'-deoxyguanosine in rat urine and liver DNA by stable isotope dilution gas chromatography/mass spectrometry.

A previously developed gas chromatographic/mass spectrometric method was applied to the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat liver DNA and in rat urine. For DNA samples, the method included: (i) fortification of samples with [15N]DNA (internal standard), (ii) enzymatic hydrolysis of DNA to deoxynucleosides, (iii) degradation of native nucleosides by treatment with trifluoroacetic acid and hydrazine, (iv) purification by C18 solid-phase extraction (SPE), (v) derivatization (acetylation followed by pentafluorobenzylation, Ac-PFB), and (vi) GC/MS analysis of the derivatives. For urine, the following methodology was used: (i) fortification of the samples with 8-18OHdG, (ii) prepurification by C18/OH SPE, (iii) derivatization, (iv) high-performance liquid chromatography purification of the Ac-PFB derivatives, and (v) GC/MS analysis. The precision of the method was demonstrated by carrying out replicate analysis of several urine and DNA samples: within-run and between-run variability was less than 5 and 8%, respectively. The analytical approaches were sufficiently sensitive to quantitate the urinary excretion of 8-OHdG (490 +/- 70 pmol/kg/24 h; sample size, 600 microliters urine) and to measure the level of 8-OHdG in liver DNA (20 8-OHdG/10(6) deoxynucleosides; sample size, 30 micrograms DNA) of rats not deliberately exposed to oxidative stress. Major advantages over previous methods are increased precision due to the use of proper isotopically labeled internal standards, and increased sensitivity due to the optimization of cleanup procedures. The simultaneous analysis of standards of three different oxidized nucleosides, namely 8-OHdG, thymidine glycol, and 5-hydroxy-methyl-2'-deoxyuridine, is shown.

Irradiated (15N)DNA as an internal standard for analysis of base-oxidized DNA constituents by isotope dilution mass spectrometry.

A simple and convenient procedure for the preparation of isotopically labeled DNA enriched in oxidized deoxynucleosides is described. 15N-Labeled DNA was isolated from Escherichia coli cells grown in an isotopically enriched medium, and the level of oxidative damage was increased by in vitro irradiation under oxygen. The resulting DNA was hydrolyzed and subsequently analyzed by GC/MS. Results indicated that the DNA was 99% 15N-enriched and that 1% of the total 2'-deoxyguanosine was converted into 8-hydroxy-2'-deoxyguanosine (8-OHdG). When applied to the analysis of 8-OHdG, [15N]DNA as internal standard gave a better reproducibility (CV, 7.9%; n = 5) as compared to the monomeric 8-[18O]hydroxy-2'-deoxyguanosine (CV, 16%; n = 4). Background levels of 8-OHdG in rat colon DNA determined with [15N]DNA and 8-18OHdG as internal standard were 26 +/- 11 and 15 +/- 7 8-OHdG per 10(6) deoxynucleosides, respectively.

Method for the analysis of oxidized nucleosides by gas chromatography/mass spectrometry.

A method for sensitive analysis of the oxidatively modified nucleosides 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU) is described. The method combines acetylation and pentafluorobenzylation of the nucleosides followed by analysis by gas chromatography/electron capture negative ion chemical ionization-mass spectrometry. The detection limits of the method for aqueous standards of HMdU and 8-OHdG were 12 and 18 fmol of starting material (signal-to-noise ratio, 3:1), respectively. The method was linear for 8-hydroxy-2'-deoxyguanosine over five orders of magnitude and gave satisfactory reproducibilities (intraassay RSD < or = 5%) for the analysis of 8-OHdG in both aqueous standards and urine fortified at the level of 35 nM. The limit of detection for the analysis of 8-OHdG in urine was 1.8 pmol, corresponding to a level of 8-OHdG in urine of 35 nM (10 micrograms/liter) at a urine sample volume of 50 microliters. Besides urine the method was applied to the analysis of 5-hydroxy-methyl-2'-deoxyuridine isolated from genomic DNA of Bacillus subtilis bacteriophage H1. Results obtained indicate that the method is potentially suitable for the determination of oxidized nucleosides in biological samples. The selectivity of the method should be enhanced in order to lower the limit of detection in biological samples.

The identification of hydroxymethyluracil in DNA of Trypanosoma brucei.

We have previously reported the detection of two unusual nucleotides, pdJ and pdV, in the DNA of Trypanosoma brucei (Gommers-Ampt et al., 1991). pdJ was found to be a novel nucleotide and is possibly involved in the regulation of variant specific surface antigen gene expression in trypanosomes. Recent evidence suggests that V could be a precursor of J, making V a key compound in the study of the biosynthesis and function of J. We have therefore determined the structure of V and here we present proof that V is HOMeU. The identity is based on a detailed comparison of dV(p) with authentic HOMedU(p), showing: I) co-migration in three different liquid chromatography analyses II) identical UV absorbance characteristics III) identical behavior in acetyl-pentafluorobenzyl derivatization and subsequent Gas chromatography/Mass spectrometry (GC/MS). The GC/MS technique has not been used before to analyse HOMedU purified from biological material. Because of its high sensitivity, it may also be useful for the detection of the low amounts of HOMedU resulting from oxidative damage of DNA.