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.     

Recultivation of Agricultural Land Impaired by Construction of a Hydropower Plant on the Sava River,Slovenia

Hydropower plants on the lower river Sava, Slovenia, were developed without sealing the underground upstream. As a consequence, without the countermeasures of elevating and recultivating, the agricultural land on the river banks would be inundated because of the water‐table increase of the river. To remedy this, the fields were elevated and recultivated. The goal of this study was to assess soil quality and production potential after land raising and recultivation and to answer the question whether it is possible to recover soil quality and crop yield after large‐scale mass manipulation, such as land raising. After recultivation and after the second year of land reuse for the two cultures grass–clover mixture and corn on two sites, Middle Pijavsko and Lower Pijavsko, soil physical and chemical characteristics and crop yields were evaluated. Mixing of topsoil with the second horizon during removal and during backfill with filling material resulted in uneven soil fertility, plant growth and decreased yield. Driving on the refilled second layer with heavy machinery caused soil compaction in the Middle Pijavsko in spite of favourable soil texture (loam, 38·7% sand). On the areas with very high sand content (over 55%) and low clay content (10%), soil compaction was not as severe or persistent. Recultivation measures restored the agricultural land almost to the production potential prior to powerplant construction. Immediate intensive land use (corn) showed less favourable effect on soil characteristics. Copyright © 2015 John Wiley & Sons, Ltd.     

A chemometric approach to the comparison of different sample treatments for metals determination by atomic absorption spectroscopy in aceto Balsamico tradizionale di Modena

A comparison of different digestion procedures has been carried out for the analysis of metal concentration in samples of vinegars and Aceto Balsamico Tradizionale of Modena (ABTM) coming from an unique barrel set. In particular, classical wet, dry ashing, and closed vessel microwave digestion procedure have been utilized and compared for each investigated species. In a few cases, direct metal determination on ABTM (without treatment procedure) is proposed as possible alternative to sample manipulation. Flame atomic absorption spectrometry was used for the quantification of iron and zinc, while graphite furnace atomic absorption spectrometry was used for all the other elements (i.e., chromium, manganese, cobalt, nickel, copper, cadmium, and lead). The comparison among the different sample treatments was carried out by the use of statistical and chemometric tools. In particular, principal component analysis and ANOVA approaches were used to discriminate between the diverse analytical methods. Furthermore, for all the dissolving techniques, the analytical metal recovery was always evaluated by the application of the recovery function on the same sample matrix. In general, the recoveries were fairly good, ranging from 90 to 103%, except for Cd and Pb with dry ashing, which showed recovery values close to 55% and 67%, respectively. As regards the metals concentration of the investigated samples, the experimental data reveal for some species the presence of concentration slightly over the legal limit fixed for wine and wine vinegar.     

Protein-lipid interactions during liposome oxidation with added anthocyanin and other phenolic compounds

Oxidation of bovine serum albumin, casein, and lactalbumin and the effect of different procyanidins, anthocyanins, and their aglycons (10 and 20 microM) on lactalbumin oxidation were investigated in a liposome system. Samples were incubated in the dark at 37 degrees C with copper, and the extent of oxidation was measured by determining the loss of tryptophan fluorescence and the formation of protein carbonyls, conjugated diene hydroperoxides, and hexanal. The correlation between different protein and lipid oxidation measurements was good and statistically significant. Casein was the most stable protein in the liposome model, and it was also the best inhibitor of liposome oxidation. All tested anthocyanins and other phenolic compounds inhibited both lipid and protein oxidation. There were no systematic differences with anthocyanins and their aglycons in relation to the concentrations used or glycosylation with either glucose or rutinose. Procyanidins B1 and B2 and ellagic acid were potentially better antioxidants than anthocyanins due to their several hydroxyl groups as measured by both protein and lipid oxidation. In conclusion, oxidative deterioration of liposomes due to protein-lipid interaction is inhibited by anthocyanins, procyanidins, and ellagitannin present, for example, in berries.     

Antioxidant activity of olive pulp and olive oil phenolic compounds of the arbequina cultivar

The aim of this study was to characterize antioxidant activities of phenolic compounds that appear in olive pulp and olive oils using both radical scavenging and antioxidant activity tests. Antiradical and antioxidant activities of olive pulp and olive oil phenolic compounds were due mainly to the presence of a 3,4-dihydroxy moiety linked to an aromatic ring, and the effect depended on the polarity of the phenolic compound. Glucosides and more complex phenolics exhibited higher antioxidant activities toward oxidation of liposomes, whereas in bulk lipids aglycons were more potent antioxidants with the exception of oleuropein. Lignans acted as antioxidants only in liposomes, which could partly be due to their chelating activity, because liposome oxidation was initiated by cupric acetate. The antioxidant activity of virgin olive oil is principally due to the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), a secoiridoid derivative (peak RT 36, structure unidentified), and luteolin.     

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair.     

The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.     

Signal-driven computations in speech processing

Learning a language requires both statistical computations to identify words in speech and algebraic-like computations to discover higher level (grammatical) structure. Here we show that these computations can be influenced by subtle cues in the speech signal. After a short familiarization to a continuous speech stream, adult listeners are able to segment it using powerful statistics, but they fail to extract the structural regularities included in the stream even when the familiarization is greatly extended. With the introduction of subliminal segmentation cues, however, these regularities can be rapidly captured.     

Identification of a DNA nonhomologous end-joining complex in bacteria

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.