Short-term and long-term effects of transient exogenous cortisol manipulation on oxidative stress in juvenile brown trout

In the wild, animals are exposed to a growing number of stressors with increasing frequency and intensity, as a result of human activities and human-induced environmental change. To fully understand how wild organisms are affected by stressors, it is crucial to understand the physiology that underlies an organism’s response to a stressor. Prolonged levels of elevated glucocorticoids are associated with a state of chronic stress and decreased fitness. Exogenous glucocorticoid manipulation reduces an individual’s ability to forage, avoid predators and grow, thereby limiting the resources available for physiological functions like the defence against oxidative stress. Using the brown trout (Salmo trutta), we evaluated the short-term (2 weeks) and long-term (4 months over winter) effects of exogenous cortisol manipulations (as well as relevant shams and controls) on the oxidative status of wild juveniles. Cortisol caused an increase in glutathione over a two-week period and appeared to reduce glutathione over winter. Cortisol treatment did not affect oxidative stress levels or low-molecular weight antioxidants. Cortisol caused a significant decrease in growth rates but did not affect predation risk. Over winter survival in the stream was associated with low levels of oxidative stress and glutathione. Thus, oxidative stress may be a mechanism by which elevated cortisol causes negative physiological consequences.</jats:p

Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol

The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis

In vitro selections of mammaglobin A and mammaglobin B aptamers for the recognition of circulating breast tumor cells

Mammaglobin B (MGB2) and mammaglobin A (MGB1) are proteins expressed in metastatic breast cancers. The early detection of circulating tumor cells (CTCs) in breast cancer patients is crucial to decrease mortality rate. Herein, novel aptamers were successfully selected and characterized agai

Facing the River Gauntlet: Understanding the Effects of Fisheries Capture and Water Temperature on the Physiology of Coho Salmon

An improved understanding of bycatch mortality can be achieved by complementing field studies with laboratory experiments that use physiological assessments. This study examined the effects of water temperature and the duration of net entanglement on physiological disturbance and recovery in coho salmon (Oncorhynchus kisutch) after release from a simulated beach seine capture. Heart rate was monitored using implanted electrocardiogram biologgers that allowed fish to swim freely before and after release. A subset of fish was recovered in respirometers to monitor metabolic recovery, and separate groups of fish were sacrificed at different times to assess blood and white muscle biochemistry. One hour after release, fish had elevated lactate in muscle and blood plasma, depleted tissue energy stores, and altered osmoregulatory status, particularly in warmer (15 vs. 10°C) and longer (15 vs. 2 min) capture treatments. A significant effect of entanglement duration on blood and muscle metabolites remained after 4 h. Oxygen consumption rate recovered to baseline within 7–10 h. However, recovery of heart rate to routine levels was longer and more variable, with most fish taking over 10 h, and 33% of fish failing to recover within 24 h. There were no significant treatment effects on either oxygen consumption or heart rate recovery. Our results indicate that fishers should minimize handling time for bycatch and maximize oxygen supply during crowding, especially when temperatures are elevated. Physiological data, such as those presented here, can be used to understand mechanisms that underlie bycatch impairment and mortality, and thus inform best practices that ensure the welfare and conservation of affected species

Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction

AbstractAlthough the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals

Cortisol predicts migration timing and success in both Atlantic salmon and sea trout kelts

Abstract Kelts – individuals of anadromous fish species which have successfully spawned and may return to sea to repeat the cycle – are perhaps the least studied life stage of iteroparous fish species. To date, our understanding of what makes them successful in their return migration to sea is limited. We investigated the relationship between three physiological parameters (baseline cortisol, baseline glucose and low molecular weight antioxidants) and the timing and success of Atlantic salmon (Salmo salar) and sea trout (Salmo trutta) kelt migration. To do so, we combined blood samples obtained within 3 minutes of capture and acoustic telemetry to track 66 salmon and 72 sea trout as they migrated out of rivers, into fjords and out at sea. We show that baseline cortisol may be a good predictor of migration success. Individuals with high baseline cortisol levels exited the river earlier but were less likely to successfully reach the sea. Similar relationships were not observed with glucose or antioxidants. We provide the first evidence to support the role of physiological status in migration success in Atlantic salmon and sea trout kelts. Our findings contribute to our understanding of the relationship between physiology and fitness in wild animals. Further, we suggest that migration timing is a trade-off between stress and readiness to migrate

The Nrf1 CNC-bZIP Protein Is Regulated by the Proteasome and Activated by Hypoxia

BACKGROUND: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. PRINCIPAL FINDINGS: We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. CONCLUSIONS: Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status

Structural characterization of peroxyl radical oxidative products of antioxidant peptides from hydrolyzed proteins

This work aimed to characterize oxidative products of five unique antioxidant peptides (P1: YFDEQNEQFR, P2: GQLLIVPQ, P3: SPFWNINAH, P4: NINAHSVVY, P5: RALPIDVL) from hydrolyzed oat proteins. Peptides were reacted with 2,2′-Azobis(2-amidinopropane) dihydrochloride, a common peroxyl radical generator. Chromatographic data showed that peptide P3 was the most oxidized (67 ± 4 %) while also displaying the most ability to scavenge radicals in the oxygen absorbance capacity assay (ORAC) with an activity of 2.16 ± 0.09 μM Trolox equivalents/μM peptide. Structural characterization using mass spectrometry showed the presence of four oxidative products of P3, three of which were mono-oxygenated and the fourth di-oxygenated. The identification of these oxidative products is new and provides an opportunity to investigate their biological function. A good correlation (r = 0.889) between the degree of oxidation and the ORAC data, demonstrates the usefulness of using oxidative peptide data to predict their radical scavenging activities