Separation of olive proteins combining a simple extraction method and a selective capillary electrophoresis (CE) approach: application to raw and table olive samples

A simple extraction method was developed to extract proteins from olive samples based on chloroform/methanol extraction followed by a protein precipitation with cold acetone. Then, a capillary electrophoresis (CE) method was carried out using an acid buffer (1 M formic acid at pH 2) to ensure a positive net charge for proteins and a neutral charge for potential interferents as polyphenols. The method developed was applied to raw and table olive samples. Interestingly, raw olive samples showed differences in protein profiles depending upon the botanical variety of olives and their geographical region. Protein profiles obtained for table olives also showed differences according to the sample treatment. Thus, a signal reduction in the electropherograms obtained for black olives was observed in comparison to those achieved for treated green olives. In this work, the use of protein profiles was demonstrated to be a powerful tool for studying variations among olive samples.     

The susceptibility of Lymnaeid snails to Paramphistomum cervi infections in Mexico

The occurrence of Paramphistomum cervi has been reported in Mexico, but its intermediate hosts have not been identified. Five species of snails of the genus Lymnaea, L. bulimoides, L. columella, L. cubensis, L. humilis and L. palustris, were collected in Mexico. Only three of the exposed species, L. palustris, L. cubensis and L. humilis, became infected with P. cervi. The first of these snail species was highly susceptible to infection and a higher number of the exposed snails shed cercariae. It is suggested that L, palustris may act as an important vector of P. cervi in Mexico.     

Establishment of proliferative cell nuclear antigen gene as an internal reference gene for polymerase chain reaction of a wide range of archival and fresh mammalian tissues.

Polymerase chain reaction (PCR) from paraffin-embedded tissues provides a powerful tool to amplify DNA from a variety of recent and archival material. Because DNA from paraffin-embedded samples is more degraded than from fresh material, the amplification of reference genes is essential to exclude false-negative results. This study describes the use of the proliferative cell nuclear antigen (PCNA) gene as a reference gene in a range of animal species and in humans. The PCNA-PCR to amplify a fragment extending from exon 5 through exon 6 and including the intervening intron 6 gave a reproducible pattern, with a 280-base pair (bp) band from canine, equine, bovine, ovine, and caprine samples showing high sequence homology. Porcine, guinea pig, tiger, and lion samples, however, gave an additional fragment of approximately 197 bp. The whole intron 6 from these fragments is missing, possibly representing a pseudogene. In feline samples only the 197-bp fragment could be detected. This study shows that the PCNA gene is highly conserved across a broad range of animal species and is well suited as an internal control for PCR analysis in veterinary medicine.     

Sunflower mutant containing high levels of palmitic acid in high oleic background

A new sunflower mutant, CAS-12, was obtained, which has both high palmitic (≈30%) and high oleic acid contents, and also a substantial amount of palmitoleic acid (≈7%). The mutant was selected after X-ray irradiation of dry seeds of the inbred line BSD-2-423, which had normal palmitic (≈3%) and high oleic (≈88%) acid levels. The increase of palmitic and palmitoleic acids occurred at the expense of the oleic acid content, which decreased to around 55% in respect to the original line. Linoleic acid content is always under 5%. Palmitic and palmitoleic acid levels were similar to those of the high palmitic mutant CAS-5 obtained in a previous programme from a low oleic line isogenic to BSD-2-423 using a similar mutagenic treatment. In that previous programme we also selected three high stearic acid mutants using chemical mutagenic treatment on the same sunflower line (RDF-1-532). We attempted to obtain mutants in other lines but were unsuccessful. The isolation of similar mutants in isogenic parental lines illustrates the importance of the genetic background in the development of specific mutants with an altered seed oil fatty acid composition. The oil of this mutant will increase the range of potential uses of sunflower oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Yield Formation in Mediterranean durum wheats under two contrasting water regimes based on path-coefficient analysis

The components of grain yield are altered by adverse growing conditions as the effects of certain environmental factors on crop growth and yield differ depending upon the developmental stages when these conditions occur. Path-coefficient analysis was used to investigate the main processes influencing grain yield and its formation under Mediterranean conditions. Twenty-five durum wheat genotypes, consisting of four Spanish commercial varieties and 21 inbred lines from the ICARDA durum wheat breeding program, were grown during 1997 and 1998 under both rainfed and irrigated conditions in southern Spain. {P}ath-coefficient analysis revealed that under favourable conditions grain yield depended in equal proportion on the three primary yield components (i.e. spikes m⁻², grains spike⁻¹, and mean grain weight), whereas in the rainfed experiments, variations in grain yield were due mainly to spikes m⁻² and to a lesser extent to grains spike⁻¹. Compensatory effects were almost absent under irrigated treatments; however, under water shortage, spikes m⁻² exerted a negative influence on grain spike⁻¹ due mainly to a negative interrelationship between tiller production and apical development. These compensatory effects could partially explain the restricted success in durum wheat breeding observed in water-limited environments of the Mediterranean region. Under rainfed conditions the number of spikes m⁻² depended mainly on the ability for tiller production, whereas in the irrigated experiments the final number of spikes was determined mostly by tiller survival.     

Fate of heavy metals in a strongly acidic shooting‐range soil: small‐scale metal distribution and its relation to preferential water flow

To assess the mobility of Pb and associated metals in a highly contaminated shooting range soil (Losone, Ticino, Switzerland), we investigated the spatial distribution of the metals and their relation to preferential water flow paths. A 2.2 m² plot located 40 m behind the stop butt was irrigated with a solution containing bromide and Brilliant Blue, a slightly sorbing dye. A soil profile 50 cm in width was sampled down to 80 cm with a spatial resolution of 2.5 cm, resulting in 626 samples. Concentrations of elements (12 ≤ Z ≤ 92) were determined by energy‐dispersive Xray fluorescence spectrometry, and Brilliant Blue concentrations were determined with a chromameter. In the acidic (pH 3), organic matter‐rich, well drained Dystric Cambisol, maximum concentrations of 80.9 g kg^‐1 Pb, 4.0 g kg^‐1 Sb, and 0.55 g kg^‐1 Cu were measured in the topsoil. Within 40 cm soil depth, however, Pb, Sb, and Cu approached background concentrations of 23 mg kg^‐1, 0.4 mg kg^‐1, and 9.4 mg kg^‐1, respectively. The even horizontal distribution and the steep gradient along soil depth indicate tight metal binding in the topsoil, and a fairly homogeneous transport front. In contrast, water flow through the profile was highly heterogeneous. In the uppermost 20 cm, preferential flow was initiated by heterogeneous infiltration at the soil surface, but had no influence on metal distribution. Below 20 cm, however, preferential flow originated from larger tree roots, and metal concentrations were significantly elevated along these macropores. Spatial distributions of Pb, Sb, and Cu were similar, suggesting that all three metals are strongly retained in the topsoil and transported along preferential water flow paths in the subsoil.     

Nitrosation of phenolic compounds: inhibition and enhancement

The nitrosation of phenol, m-, o-, and p-cresol, 2,3-, 3,5-, and 2, 6-dimethylphenol, 3,5-di-tert-butylphenol, 2,4,6-trimethylphenol, o-chlorophenol, and o-bromophenol was studied. Kinetic monitoring of the reactions was accomplished by spectrophotometric analysis of the products at 345 nm. At pH > 3, the dominant reaction was C-nitrosation through a mechanism that appears to consist of an attack on the nitrosatable substrate by NO(+)/NO(2)H(2)(+), followed by a slow proton transfer. The finding of an isokinetic relationship supports the idea that the same mechanism operates throughout the series. The observed sequence of nitrosatable substrate reactivities is explained by (i) the preferred para-orientation of the hydroxyl group for the electrophilic attack of nitrosating agents, (ii) steric hindrance of alkyl substituents, which reduces or prevents attack by nitrosating agents, and (iii) the hyperconjugative effect of the methyl substituent, which causes electronic charge to flow into the aromatic nucleus, as well as the opposite electronic withdrawing effect induced by halogen substituents. The results show that potential nitrosation of widespread environmental species such as chlorophenols is negligible, but more attention should be paid to polyphenols with strongly nucleophilic carbon atoms.     

Influence of micelles on the basic degradation of carbofuran

The effect of micellar aggregates upon the stability of carbofuran in basic media has been studied. The effect of the presence of micelles upon the basic hydrolysis of carbofuran is a function of the nature of the surfactant monomer. Important catalysis of basic hydrolysis of carbofuran in the presence of colloid aggregates with positive surface charge has been reported. On the other hand, the presence of anionic and nonionic surfactants implies a large inhibition of the basic hydrolysis of carbofuran. Both catalysis and inhibition are due to the association of carbofuran with the micellar core. The kinetic constants for the basic hydrolysis of carbofuran in these microheterogeneous media have been obtained on the basis of a micellar pseudophase model. No significant changes in the intrinsic reactivity of HO- against carbofuran have been observed.