Fish proteins as targets of ferrous-catalyzed oxidation: identification of protein carbonyls by fluorescent labeling on two-dimensional gels and MALDI-TOF/TOF mass spectrometry

Protein oxidation in fish meat is considered to affect negatively the muscle texture. An important source of free radicals taking part in this process is Fenton's reaction dependent on ferrous ions present in the tissue. The aim of this study was to investigate the susceptibility of cod muscle proteins in sarcoplasmic and myofibril fractions to in vitro metal-catalyzed oxidation and to point out protein candidates that might play a major role in the deterioration of fish quality. Extracted control proteins and proteins subjected to free radicals generated by Fe(II)/ascorbate mixture were labeled with fluorescein-5-thiosemicarbazide (FTSC) to tag carbonyl groups and separated by two-dimensional gel electrophoresis. Consecutive visualization of protein carbonyl levels by capturing the FTSC signal and total protein levels by capturing the SyproRuby staining signal allowed us to quantify the relative change in protein carbonyl levels corrected for changes in protein content. Proteins were identified using MALDI-TOF/TOF mass spectrometry and homology-based searches. The results show that freshly extracted cod muscle proteins exhibit a detectable carbonylation background and that the incubation with Fe(II)/ascorbate triggers a further oxidation of both sarcoplasmic and myofibril proteins. Different proteins exhibited various degrees of sensitivity to oxidation processes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), nucleoside diphosphate kinase B (NDK), triosephosphate isomerase, phosphoglycerate mutase, lactate dehydrogenase, creatine kinase, and enolase were the sarcoplasmic proteins most vulnerable to ferrous-catalyzed oxidation. Moreover, NDK, phosphoglycerate mutase, and GAPDH were identified in several spots differing by their pI, and those forms showed different susceptibilities to metal-catalyzed oxidation, indicating that post-translational modifications may change the resistance of proteins to oxidative damage. The Fe(II)/ascorbate treatment significantly increased carbonylation of important structural proteins in fish muscle, mainly actin and myosin, and degradation products of those proteins were observed, some of them exhibiting increased carbonylation levels.     

Distribution of Cd, Ni, Cr, and Pb in Amended Soils from Alicante Province (SE, Spain)

Heavy metals can be transferred from soils to other ecosystem parts and affect ecosystems and human health through the food chain. Today the use of biosolids to improve the nutrient contents of a soil is a common practice. Contamination of soils by potentially toxic elements (e.g., Cd, Ni, Cr, Pb) from amendments of biosolids is subject to strict controls within the European Community in relation to total permissible metal concentrations, soil properties and intended use. In this context, heavy metal concentrations were studied in agricultural soils devoted to vegetable crops in the province of Alicante (SE Spain), where an intensive agriculture takes place. This study is aimed at ascertaining the chemical partitioning of Cd, Ni, Cr, and Pb in agricultural soils repeatedly amended with sludge. Selected soil properties relevant to control the mobility and bioavaibility of heavy metals were analyzed for the general characterisation of these agricultural soils. The distribution of chemical forms of Cd, Ni, Cr, and Pb in five biosolids-amended soils was studied using a sequential extraction procedure that fractionates the metal into soluble-exchangeable, specifically sorbed-carbonate bound, oxidizable, reducible, and residual forms. The biosolids incorporation has modified the soil composition, leading to the increment of heavy metals. The residual, reducible, and carbonate-sorbed forms were dominant. Detailed knowledge of the soil at the application site, especially pH, CEC, buffering capacity, organic matter, clay minerals, and clay content, is essential.     

Separation and HPLC-MS identification of phenolic antioxidants from agricultural residues: almond hulls and grape pomace

Almond hulls and grape pomace are residues abundantly generated by agricultural industries, which could be processed to obtain bioactive products. To this purpose, crude ethanol extracts from both agricultural byproducts were attained and subsequently fractionated in order to obtain an organic/water fraction (FOW). Extracts and fractions were analyzed for antioxidant power and their phenolic components tentatively identified by HPLC-MS. Chromatographic peaks of almond hull extracts showed the occurrence of hydroxybenzoic and cinnamic acid derivatives, with minor presence of flavan-3-ols (ECG, EGCG), whereas the FOW fraction offered the additional presence of epicatechin (EC) and glycosylated flavonols. In the composition for extracts of white and red grape pomace several of these compounds were also detected but basically consisted of glycosylated flavonols (quercetin, kaempferol). As a difference between both grape pomaces, myricetin glycosyde was found in that from the red variety, whereas flavan-3-ols (EC, afzelechin) were only identified in white pomace. When their FOW fractions were analyzed, gallic acid and some hydroxybenzoic acids were additionally detected. Antioxidant activity was assessed by DPPH and TBARS assays. Almond hulls showed inhibition percentages lower than 50% in both assays, while the inhibition percentage ranged from 80% to 90% in pomace extracts. Red grape pomace extract was the most efficient antioxidant, with an EC50 value of 0.91 g/L for TBARS and 0.20 g/L for DPPH. Even appearing as two quite different vegetal matrixes, the composition of phenolics in grape pomace and almond hulls is quite similar, the main difference being the major occurrence of flavonols in grape pomace. This fact could presumably explain the lower antiradical activity of hull extracts.     

Plant availability of heavy metals in a soil amended with a high dose of sewage sludge under drought conditions

The objective of this research was to study the effect of water deficit on soil heavy metal availability and metal uptake by ryegrass (Lolium multiflorum Lam.) plants grown in a soil amended with a high dose of rural sewage sludge. Three fertility treatments were applied: sewage sludge (SS), mineral fertilizer (M), and control (C); unamended). The levels of irrigation were: well-watered (W) and water deficit (D). Microbial respiration decreased the total organic C (TOC) in sludge-treated soils, but this did not enhance soil DTPA-extractable heavy metal concentrations. Indeed, Zn, Cu, Mn and Ni availability decreased during the experiment. C- and M-treated soils showed either no changes or increases of some trace element concentrations during the incubation. In the plant experiment, ryegrass dry matter (DM) yield, relative water content (RWC) and leaf water potential (_w) decreased in drought conditions. Sludge addition increased metal concentrations in plants. However, in some instances, SS-treated plants showed either similar or lower transfer coefficient (Tc) values than did plants in the C and M treatments. Water deficit decreased the concentration and the Tc of some metals in roots of M and SS plants. Results indicate that sludge-borne heavy metals were maintained in chemical forms of low availability. The lower metal uptake by SS and M plants under dry conditions cannot be attributed to a lower availability of these elements in soil.     

Transport of commercial endosulfan through a column of aggregated vineyard soil by a water flux simulating field conditions

Endosulfan is a potentially harmful, degradation resistant pesticide that is found in soils where it has been used. Despite being hydrophobic and having high affinity for soil matrix components, it has also been found in groundwater. To investigate this behavior, we studied the passage of a commercial emulsified formulation through a column of aggregated vineyard soil under simulated light rain. Breakthrough data were obtained using gas chromatography with electron capture detection to determine the concentration of endosulfan in samples extracted from the column periodically at several depths. These data, and analogous data obtained previously for the passage of bromide, were analyzed using the program CXTFIT v.2. Analysis of the bromide data strongly suggested the existence of preferential flow paths in the column. The endosulfan data were adequately accounted for by a model in which the preferential flow and nonpreferential flow regions are almost isolated from each other. These regions differ also as regards both the partition of endosulfan between soil and soil solution and the rate at which reversibly adsorbed endosulfan is transformed into irreversibly adsorbed endosulfan. The "irreversibility" sink term accounts also for biological and chemical degradation of endosulfan. The findings imply that soil humidity favors the transport of commercial endosulfan by the formation and maintenance of preferential flow paths in soil, controlling both the presence of endosulfan in groundwater and its high persistence in soil.     

Influence of thermal treatments simulating cooking processes on the polyphenol content in virgin olive oil

Virgin olive oils were subjected to simulated common domestic processing, including frying, microwave heating, and boiling with water in a pressure cooker. The impact of these processes on polyphenol content and physicochemical characteristics of oils was assessed. Thermal oxidation of oils at 180 degrees C caused a significant decrease in hydroxytyrosol- and tyrosol-like substances. In contrast, oils heated for 25 h still retained a high proportion of the lignans 1-acetoxypinoresinol and pinoresinol. Thermal oxidation also resulted in a rapid degradation of alpha-tocopherol and the glyceridic fraction of oils. Microwave heating of oils for 10 min caused only minor losses in polyphenols, and the oil degradation was lower than that in thermoxidation assays. Again, lignans were the least affected polyphenols and did not change during microwave heating. Boiling a mixture of virgin olive oil and water in a pressure cooker for 30 min provoked the hydrolysis of the secoiridoid aglycons and the diffusion of the free phenolics hydroxytyrosol and tyrosol from the oil to the water phase. Losses of polyphenols were detected only at pH lower than 6. Moreover, alpha-tocopherol and the glyceridic fraction of oils were not modified during this process. It is worth noting that all the heating methods assayed resulted in more severe polyphenols losses and oil degradation for Arbequina than for Picual oil, which could be related to the lower content in polyunsaturated fatty acids of the latter olive cultivar. These findings may be relevant to the choice of cooking method and olive oil cultivar to increase the intake of olive polyphenols.     

New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing

Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.     

浙西北天然次生林群落物种多样性研究

对浙西北地区天然次生林群落植物物种多样性特征进行了研究,测定了群落植物生长型构成以及各层次植物物种的重要值,采用多种测度方法计算了群落各层次植物物种的丰富度指数、均匀度指数和多样性指数。结果表明：（1）浙西北地区天然次生林群落各层次优势种比较明显,乔木树种以青冈为主,灌木树种以柃木和檐木占优势,草本植物则以箬竹为主;（2）该地区天然次生林群落植物物种比较丰富,乔木树种种数最多,草本植物稀少;就各层次而言,草本层物种数最多,乔木层和灌木层比较接近,各层次的均匀度指数的趋势是灌木层最高,草本层最低,乔木层居中;以多种指标计算各层次物种多样性指数结果均显示,灌木层多样性指数最高,草本层最低,尽管草本层具有最高的丰富度;（3）封育时间长、人为干扰轻的地区具有较高的物种多样性指标以及数量指标,有利于生物多样性的保护。     

Plants and biological soil crusts modulate the dominance of N forms in a semi-arid grassland

It has been suggested that the dominance of N forms should shift from dissolved organic nitrogen (DON) to nitrate along a gradient of increasing N availability. We aimed to apply this model at a local scale within a semi-arid ecosystem showing a high spatial heterogeneity in the distribution of vegetation and soil resources. By doing this, we seek a better understanding of the N cycling in spatially heterogeneous ecosystems. We took soil samples from the three major sources of spatial heterogeneity: the grass Stipa tenacíssima, the N-fixing shrub Retama sphaerocarpa, and open areas. We also sampled the biological soil crust (BSC) located in the latter areas as another source of spatial heterogeneity. BSC microsites were classified by four levels of soil coverage, ranging from high coverage (66%) to bare soil. The proportion of nitrate, ammonium and DON was determined in all microsites. DON was the dominant N form for open areas, while nitrate was dominant under the canopy of Retama; these microsites contained the lowest and highest N availability, respectively. Under BSC, DON was the dominant N form. We found high temporal variability in the dominance of N forms for all microsites. Our results suggest that the biome-derived model of Schimel and Bennett (2004) explaining N form dominance across N availability gradients may be extended to local gradients.     

Removal of Nickel by Means of Micellar-Enhanced Ultrafiltration (MEUF) Using Two Anionic Surfactants

Micellar-enhanced ultrafiltration (MEUF) is an effective separation technique for removing metal ions from aqueous environments. The critical micellar concentration (CMC) of two anionic surfactants, sodium dodecyl sulfate (SDS) and linear alkylbenzene sulfonate (LAS), was determined by means of conductometry. The effects of pH, conductivity, and surfactant concentration on the permeate flow, retention of surfactants and nickel by MEUF, was studied. Results showed that for surfactant concentrations beyond the CMC, Ni(II) retention with SDS was slightly higher than with LAS (S/M?=?45: Ni(II) retention was 70% and 55% for SDS and LAS, respectively). LAS surfactant was always retained in higher quantities than SDS. An increase in conductivity produced large reduction in Ni(II) retention and slightly increased surfactant retention. pH values between 4 and 8 did not affect nickel retention but enhanced the SDS and LAS surfactant retentions.