An automated flow system for in-line extraction of proteins and separation of jacalin using a low-cost stationary phase.

Lectins are carbohydrate binding proteins with many physiological and biotechnological applications. Isolation of proteins is normally time-consuming and encompasses multiple and, sometimes, complicated steps that hinder reproducibility and yield. Affinity chromatography is an efficient way to simplify and improve protein purification, however often requiring an expensive and fragile stationary phase. In this regard, automated flow-based systems minimize the time for extraction of species from solid samples without hindering the features of batch procedures. In this work, a new inexpensive affinity-based stationary phase was developed for in-line separation of jacalin, a galactose-binding lectin from jackfruit seeds. In the flow manifold, in-line extraction of proteins was also carried out with continuous monitoring using the spectrophotometric Biuret assay. For protein determination, linear response was observed from 3.0 to 15 g L-1. The results of the analysis of protein extracts from jackfruit seeds obtained with the herein described procedure and batch procedure agreed with 95% confidence level. Quantitative extraction of proteins from jackfruit seed powder required recirculation of extraction buffer for 15 min through a lab-made polymethylmethacrylate (PMMA) column containing 200 mg of the crude seed powder. In the chromatographic step, jacalin was isolated after 300 s. Therefore, three essential steps for jacalin isolation were performed in one manifold in a fast way, minimizing sample consumption and solution handling. Additionally, the versatile and multi-task developed flow manifold can be useful for routine analysis and preparative procedures, being adaptable for the extraction and separation of other species from solid matrixes with continuous monitoring of the processes.

Gene expression and biochemical responses in brain of zebrafish Danio rerio exposed to organic nanomaterials: carbon nanotubes (SWCNT) and fullerenol (C60(OH)18-22(OK4)).

Nanomaterials (NM) industry had grown in the last decade, although there are few studies concerning its potential toxicity effects on aquatic organisms. In this study the freshwater zebrafish (Danio rerio) was exposed to two kinds of carbon NM, single-wall carbon nanotubes (SWCNT) and fullerenol [C60(OH)18-22(OK4)] to analyze oxidative stress responses on fish brain. Adult zebrafish (mean mass: 0.52±0.01g) were submitted to intraperitoneal injections of SWCNT suspension and fullerenol solution (30mg/kg of fish), receiving one or two doses with a time interval of 24h. Results showed that total antioxidant capacity was lowered in brains of fish exposed 24h to fullerenol when compared to those from SWCNT treatment (p<0.05). After 48h, fullerenol induced higher expression of both catalytic and regulatory subunits of enzyme glutamate cysteine ligase when compared to control group (p<0.05), indicating an antioxidant behavior. In vitro assays showed a dual effect of SWCNT, since a pro-oxidant behavior was observed at low concentrations (0.1 and 1.0mg/L) and an antioxidant one at the highest concentration (10.0mg/L). Few biological responses were altered by this NM: decrease in total antioxidant capacity and induction of the expression of the transcription factor Nrf2 when compared to control group.

Biochemical and behavioral responses in the estuarine polychaete Perinereis gualpensis (Nereididae) after in situ exposure to polluted sediments.

In situ translocation experiments are advantageous relative to traditional laboratory experiments, particularly for understanding the bioavailability of trace metals like mercury (Hg). Individuals of the polychaete Perinereis gualpensis were translocated from a reference site (Raqui estuary, Chile) to an estuarine site with significant sediment Hg concentrations (Lenga estuary: 1.78-9.89 mg/kg). Individuals were exposed in polluted and non-polluted sediments for 21 days and sampled every 7 days with cages deployed at three different depths. Tissue Hg concentrations were measured in conjunction with oxidative stress responses. Translocated polychaetes rapidly accumulated Hg. Glutathione S-transferase (GST) activities measured from posterior body regions were 2-fold higher than control activities after 21 days of exposure. Other antioxidant measures were idiosyncratic. Distinct burrowing behavior differences were observed; control polychaetes exhibited more homogenous vertical distributions, whereas in Lenga, worms tended to remain in upper layers. These studies demonstrate that under natural conditions, Hg is highly bioavailable to polychaetes affecting both biochemical and behavioral responses after relatively short-term exposure.

Evaluation of the antioxidant capacity of synthesized coumarins.

Coumarins are secondary metabolites that are widely distributed within the plant kingdom, some of which have been extensively studied for their antioxidant properties. The antioxidant activity of coumarins assayed in the present study was measured by different methods, namely the 1,1-diphenyl-2-picryl-hydrazyl (DPPH(•)) method, cyclic voltammetry and the antioxidant capacity against peroxyl radicals (ACAP) method. The 7,8-dihydroxy-4-methylcoumarin (LaSOM 78), 5-carboxy-7,8-dihydroxy-4-methylcoumarin (LaSOM 79), and 6,7-dihydroxycoumarin (Esculetin) compounds proved to be the most active, showing the highest capacity to deplete the DPPH radicals, the highest antioxidant capacity against peroxyl radicals, and the lowest values of potential oxidation.

Effects of carbon nanomaterials fullerene C60 and fullerol C60(OH)18₋22 on gills of fish Cyprinus carpio (Cyprinidae) exposed to ultraviolet radiation.

In consequence of their growing use and demand, the inevitable environmental presence of nanomaterials (NMs) has raised concerns about their potential deleterious effects to aquatic environments. The carbon NM fullerene (C60), which forms colloidal aggregates in water, and its water-soluble derivative fullerol (C60(OH)18₋22), which possesses antioxidant properties, are known to be photo-excited by ultraviolet (UV) or visible light. To investigate their potential hazards to aquatic organisms upon exposure to UV sunlight, this study analyzed (a) the in vitro behavior of fullerene and fullerol against peroxyl radicals (ROO) under UV-A radiation and (b) the effects of these photo-excited NMs on oxidative stress parameters in functional gills extracted from the fish Cyprinus carpio (Cyprinidae). The variables measured were the total antioxidant capacity, lipid peroxidation (TBARS), the activities of the antioxidant enzymes glutathione reductase (GR) and glutamate cysteine ligase (GCL), and the levels of the non-enzymatic antioxidant glutathione (GSH). The obtained results revealed the following: (1) both NMs behaved in vitro as antioxidants against ROO in the dark and as pro-oxidants in presence of UV-A, the latter effect being reversed by the addition of sodium azide, which is a singlet oxygen (¹O2) quencher; (2) fullerene induced toxicity with or without UV-A incidence, with a significant (p<0.05) increase in lipid peroxidation (with greater damage under illumination), a decrease in GCL activity, and the depletion of GSH stocks (under illumination), all of which were attributed to ¹O2 generation; and (3) fullerol also decreased GCL activity and GSH formation (p<0.05) but without lipid damage. The overall results show that fullerene can be toxic with or without light incidence, whereas UV radiation seems to play a key role in the environmental toxicity of carbon NMs through ¹O2 formation.

Multibiomarker approach at different organization levels in the estuarine Perinereis gualpensis (Polychaeta; Nereididae) under chronic and acute pollution conditions.

This work aimed to study biochemical biomarkers and population responses in the polychaete Perinereis gualpensis (Nereididae) from two Chilean estuaries, Lenga (36°45'S; 73°10'W) and Tubul-Raqui (37°14'S,73°26'W). Lenga estuary is characterized by high mercury and PAH levels as consequence of several historic incidental spills while Tubul is mainly impacted by intensive fish activity. Raqui estuary, with low anthropogenic impact was considered the reference site. Worms were collected in winter and summer periods and tissues were differentiated between anterior (first 40 segments) and posterior region (the rest of the body). Levels of the antioxidant glutathione (GSH), glutathione-S-transferase (GST) activity, total antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (TBARS) levels, Na+, K+-ATPase activity (NKA) and metalothioneins (MTs) content were determined in each region. L3 (combined length of prostomiun+peristomiun+first segment) length, size, frequency, histograms and density of individuals were studied in all sites and seasons. Significant differences (p<0.05) were observed among sites for most of the biochemical responses (GSH, GCL, GST, ACAP and TBARS), showing variation between seasons and body regions. Population responses in the most polluted estuary (Lenga) showed differences in size, frequency, lengths and low densities compared with lower impacted sites, indicating the costs associated with tolerance in organisms chronically exposed to estuarine pollution.