Thiol Oxidation Research Articles

Thiol-disulfide balance and trace element levels in patients with seasonal allergic rhinitis.

The prevalence of allergic diseases is gradually increasing worldwide. The most common such allergic disease is allergic rhinitis (AR). The present study investigated the possible relationship between seasonal AR and the thiol-disulfide balance and zinc and copper levels in adult individuals. 130 male and female adults were included in the study. The participants' serum thiol-disulfide balance and zinc and copper levels were measured spectrophotometrically using commercial kits. Statistical significance was accepted as p < 0.05 between the groups. The serum copper (p = 0.001), native thiol (p = 0.006), reduced thiol (p < 0.001), and thiol oxidation reduction ratio (p < 0.001) levels were significantly lower in the seasonal AR group than in the control group. In AR patients, the low level of copper, which is an important trace element, the deterioration of the thiol-disulfide balance, which represents a unique indicator of the oxidant-antioxidant balance, the increased disulfide level caused by oxidative stress, and the decreased native thiol level can all serve as important biochemical markers.

Enzymatically Triggered Deprotection and Cross-Linking of Thiolated Alginate-Based Bioinks

Thiolated polymers are widely used in hydrogels for drug delivery, tissue engineering, and biofabrication. The oxidation of thiols is spontaneous, resulting in the formation of disulfide bridges and cross-linking of polymers. The cross-linking process is, however, difficult to control and is initiated directly when the thiolated components are exposed to ambient conditions, which significantly complicates handling of the materials. Here, we show a fully bioorthogonal enzyme-mediated thiol-based chemistry for dynamic covalent cross-linking of carbohydrate-based hydrogels that circumvents the problems with uncontrolled thiol oxidation. Alginate was modified with cysteine residues, protected by an enzyme-labile thiol-protecting group (Phacm). Releasing the Phacm group by penicillin G acylase generates free thiols that oxidize under physiological conditions, resulting in a reversible cross-linking and formation of hydrogels with tunable stiffness. Prior to deprotection, the components can be exposed to ambient conditions. The enzyme-triggered deprotection and subsequent gelation allows for encapsulation of cells and 3D bioprinting of cell-laden hydrogel structures. Remaining deprotected thiols enabled postprinting modifications and hydrogel self-healing. The proposed hydrogel synthesis strategy significantly increases the versatility of thiol-based cross-linking chemistries and provides new possibilities to generate dynamic covalent hydrogels for a broad range of biomedical applications.

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation?

Metal and redox homeostasis in cyanobacteria is tightly controlled to preserve the photosynthetic machinery from mismetallation and minimize cell damage. This control is mainly taken by FUR (ferric uptake regulation) proteins. FurC works as the PerR (peroxide response) paralog in Anabaena sp. PCC7120. Despite its importance, this regulator remained poorly characterized. Although FurC lacks the typical CXXC motifs present in FUR proteins, it contains a tightly bound zinc per subunit. FurC: Zn stoichiometrically binds zinc and manganese in a second site, manganese being more efficient in the binding of FurC: Zn to its DNA target PprxA. Oligomerization analyses of FurC: Zn evidence the occurrence of different aggregates ranging from dimers to octamers. Notably, intermolecular disulfide bonds are not involved in FurC: Zn dimerization, dimer being the most reduced form of the protein. Oligomerization of dimers occurs upon oxidation of thiols by H2O2 or diamide and can be reversed by 1,4-Dithiothreitol (DTT). Irreversible inactivation of the regulator occurs by metal catalyzed oxidation promoted by ferrous iron. However, inactivation upon oxidation with H2O2 in the absence of iron was reverted by addition of DTT. Comparison of models for FurC: Zn dimers and tetramers obtained using AlphaFold Colab and SWISS-MODEL allowed to infer the residues forming both metal-binding sites and to propose the involvement of Cys86 in reversible tetramer formation. Our results decipher the existence of two levels of inactivation of FurC: Zn of Anabaena sp. PCC7120, a reversible one through disulfide-formed FurC: Zn tetramers and the irreversible metal catalyzed oxidation. This additional reversible regulation may be specific of cyanobacteria.

Dual cocatalysts decorated CdS nanoparticles for efficient dehydrocoupling of thiols into disulfides

Simultaneously utilizing photoinduced electrons and holes to direct splitting of thiols into value-added disulfides and clean hydrogen (H2) fuels conforms the development standards of green chemistry. Herein, a cobalt phosphate (Co-Pi) and reduced graphene oxide (GR) modified CdS photocatalyst is synthesized and utilized for photocatalytic selective conversion of 4-methoxythiophenol (4-MTP) to bis(4-methoxyphenyl) disulfide (4-MPD) coupled with H2 evolution. In this dual-cocatalyst system, Co-Pi effectively captures and releases the holes from CdS through the chemical state change of Co to accelerate the surface thiols oxidation reaction, while GR is used as an electron collector and an active site for the proton reduction to H2. Consequently, the bifunctional GR-CdS-(Co-Pi) photocatalyst demonstrates significantly higher photoactivity than blank CdS. This work affords a new paradigm of incorporating dual-cocatalysts with semiconductor photocatalysts to efficiently take advantage of the photoinduced electrons and holes for photoredox-catalyzed cooperative coupling of organic transformation and H2 evolution.

Differences in the Formation of Reactive Oxygen Species and Their Cytotoxicity between Thiols Combined with Aqua- and Cyanocobalamins.

Cobalamin is an essential nutrient required for the normal functioning of cells. Its deficiency can lead to various pathological states. Hydroxocobalamin (HOCbl) and cyanocobalamin (CNCbl) are the forms of vitamin B12 that are most commonly used for supplementation. There is substantial evidence indicating that cobalamins can both suppress and promote oxidative stress; however, the mechanisms underlying these effects are poorly understood. Here, it was shown that the oxidation of thiols catalyzed by HOCbl and CNCbl is accompanied by reactive oxygen species (ROS) production and induces, under certain conditions, oxidative stress and cell death. The form of vitamin B12 and the structure of thiol play a decisive role in these processes. It was found that the mechanisms and kinetics of thiol oxidation catalyzed by HOCbl and CNCbl differ substantially. HOCbl increased the rate of oxidation of thiols to a greater extent than CNCbl, but quenched ROS in combination with certain thiols. Oxidation catalyzed by CNCbl was generally slower. Yet, the absence of ROS quenching resulted in their higher accumulation. The aforementioned results might explain a more pronounced cytotoxicity induced by combinations of thiols with CNCbl. On the whole, the data obtained provide a new insight into the redox processes in which cobalamins are involved. Our results might also be helpful in developing new approaches to the treatment of some cobalamin-responsive disorders in which oxidative stress is an important component.

Metallic Porphyrazine Networks: Synthesis, as Well as Thermal and Optical Properties for Accelerating the Oxidation of Thiols to Their Disulfides

A condensation reaction of 2,3,5,6-tetraamino-1,4-benzoquinone 1 with 4,5-Dichloro-3,6-dihydroxy-phthalonitrile 2 produced p-benzoquinone [2,3-b:2,3-b]bis[(5,8-dihydroxybenzopyrazine)-6,7-dinitrile] 3. Utilizing acetic acid with lithium/pentanol, the tetra-nitrile monomer was cyclo-tetramerized, yielding the matching network polymer, tetra p-benzoquinone[2,3-b:2,3-b]. bis[(5,8-dihydroxybenzopyrazino) porphyrazine (2H-Pz) 4a. The equivalent tetra p-benzoquinone[2,3-b:2,3-b]bis[(5,8-dihydroxybenzopyrazino) metallic porphyrazine networks (M-Pz) M = Zn 4b or Ni 4c, were obtained by cyclo-tetramerizing the tetra-nitril monomer 3 using metal salt and quinoline. The synthesized molecules’ elemental analytical results, as well as their IR and NMR spectral data, are consistent with their assigned structures. The prepared compounds have large molecular weights and metal content, indicating that reactions of tetramerization, polymerization, and chelation were all productive. The synthesized porphyrazines were proved to be excellent substrates for oxidizing thiophenol and benzyl thiol to their respective disulfides in atmospheric oxygen. The maximal production of the corresponding disulfides after 15 min was 96 percent for thiophenol and 93 percent for benzyl thiol, respectively.

The Anti-Fungal Activity of Nitropropenyl Benzodioxole (NPBD), a Redox-Thiol Oxidant and Tyrosine Phosphatase Inhibitor.

Phylogenetically diverse fungal species are an increasing cause of severe disease and mortality. Identification of new targets and development of new fungicidal drugs are required to augment the effectiveness of current chemotherapy and counter increasing resistance in pathogens. Nitroalkenyl benzene derivatives are thiol oxidants and inhibitors of cysteine-based molecules, which show broad biological activity against microorganisms. Nitropropenyl benzodioxole (NPBD), one of the most active antimicrobial derivatives, shows high activity in MIC assays for phylogenetically diverse saprophytic, commensal and parasitic fungi. NPBD was fungicidal to all species except the dermatophytic fungi, with an activity profile comparable to that of Amphotericin B and Miconazole. NPBD showed differing patterns of dynamic kill rates under different growth conditions for Candida albicans and Aspergillus fumigatus and was rapidly fungicidal for non-replicating vegetative forms and microconidia. It did not induce resistant or drug tolerant strains in major pathogens on long term exposure. A literature review highlights the complexity and interactivity of fungal tyrosine phosphate and redox signaling pathways, their differing metabolic effects in fungal species and identifies some targets for inhibition. A comparison of the metabolic activities of Amphotericin B, Miconazole and NPBD highlights the multiple cellular functions of these agents and the complementarity of many mechanisms. The activity profile of NPBD illustrates the functional diversity of fungal tyrosine phosphatases and thiol-based redox active molecules and contributes to the validation of tyrosine phosphatases and redox thiol molecules as related and complementary selective targets for antimicrobial drug development. NPBD is a selective antifungal agent with low oral toxicity which would be suitable for local treatment of skin and mucosal infections.

The Use of Probiotics Combined with Exercise Affects Thiol/Disulfide Homeostasis, an Oxidative Stress Parameter.

Background: Intestinal microbiota play a role in the health and performance of athletes, and can be influenced by probiotics. Thus, in this study, we aimed to investigate the effect of the use of probiotics combined with chronic exercise on the thiol/disulfide homeostasis, a novel marker of oxidative stress. Methods: Male Wistar rats were randomly divided into four groups: control (Cn), exercise (Ex), probiotics (P), and probiotics + exercise (PEx). A capsule containing 6 × 108 CFU of L. rhamnosus, L. paracasei, L. acidophilus, and B. lactis was given daily for eight weeks to all the experimental animals. The total thiol (TT, μmol/L) and native thiol (NT, μmol/L) concentrations were measured to determine the oxidative stress parameters. The dynamic disulfide (DD, %), reduced thiol (RT, %), oxidized thiol (OT, %), and thiol oxidation reduction (TOR, %) ratios were analyzed. Results: The TT level was found to be significantly higher in the Ex group (p = 0.047, η2 = 0.259). The DD level, a marker of oxidation, was significantly lower in the PEx group (p = 0.042, η2 = 0.266); the highest value of this parameter was found in the Ex group. The use of probiotics alone had no effect on thiol/disulfide homeostasis. Conclusions: We showed, for the first time, that probiotics administered “with exercise” decreased dynamic disulfide and significantly reduced oxidative damage. Therefore, we speculate that the use of probiotics in sports involving intense exercise might be beneficial to reduce oxidative stress.

Carbendazim toxicity in different cell lines and mammalian tissues.

The extensive production and use of harmful pesticides in agriculture to improve crop yield has raised concerns about their potential threat to living components of the environment. Pesticides cause serious environmental and health problems both to humans and animals. Carbendazim (CBZ) is a broad spectrum fungicide that is used to control or effectively kill pathogenic microorganisms. CBZ is a significant contaminant found in food, soil and water. It exerts immediate and delayed harmful effects on humans, invertebrates, aquatic animals and soil microbes when used extensively and repeatedly. CBZ is a teratogenic, mutagenic and aneugenic agent that imparts its toxicity by enhancing generation of reactive oxygen species generation. It elevates the oxidation of thiols, proteins and lipids and decreases the activities of antioxidant enzymes. CBZ is cytotoxic causing hematological abnormalities, mitotic spindle deformity, inhibits mitosis and alters cell cycle events which lead to apoptosis. CBZ is known to cause endocrine-disruption, embryo toxicity, infertility, hepatic dysfunction and has been reported to be one of the leading causes of neurodegenerative disorders. CBZ is dangerous to human health, the most common side effects upon chronic exposure are thyroid gland dysfunction and oxidative hepato-nephrotoxicity. In mammals, CBZ has been shown to disrupt the antioxidant defense system. In this review, CBZ-induced toxicity in different cells, tissues and organisms, under in vitro and in vivo conditions, has been systematically discussed.

Copper-Catalyzed Glutathione Oxidation is Accelerated by the Anticancer Thiosemicarbazone Dp44mT and Further Boosted at Lower pH

Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, CuIn(GS)m clusters are formed, in which CuI is very slowly reoxidized by O2 at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by CuII was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that CuII-Dp44mT catalyzes GSH oxidation faster than CuII alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favors the rate-limiting reduction step without subsequent dissociation of the CuI intermediate. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of CuII-Dp44mT.

Chemoselective Oxidation of Thiols with Oxoammonium Cations.

The oxidation of various aryl and aliphatic thiols with the commercially available and environmentally benign reagent Bobbitt's salt (1) has been investigated. The reaction affords the corresponding disulfide products in good to excellent yields (71-99%) and can be accomplished in water, methanol, or acetonitrile solvent. Moreover, the process is highly chemoselective, tolerating traditionally oxidation-labile groups such as free amines and alcohols. Combined experimental and computational studies reveal that the oxidation takes place via a polar two-electron process with concomitant and unexpected deoxygenation of the oxoammonium cation through homolysis of the weak N-O bond, differing from prototypical radical-based thiol couplings. This unusual consumption of the oxidant has significant implications for the development of new nitroxide-based radical traps for probing S-centered radicals, the advancement of new electrochemical or catalytic processes involving nitroxide/oxoammonium salt redox couples, and applications to biological systems.

Reversible Thiol Oxidation Increases Mitochondrial Electron Transport Complex Enzyme Activity but Not Respiration in Cardiomyocytes from Patients with End-Stage Heart Failure.

Cardiomyocyte dysfunction in patients with end-stage heart failure with reduced ejection fraction (HFrEF) stems from mitochondrial dysfunction, which contributes to an energetic crisis. Mitochondrial dysfunction reportedly relates to increased markers of oxidative stress, but the impact of reversible thiol oxidation on myocardial mitochondrial function in patients with HFrEF has not been investigated. In the present study, we assessed mitochondrial function in ventricular biopsies from patients with end-stage HFrEF in the presence and absence of the thiol-reducing agent dithiothreitol (DTT). Isolated mitochondria exposed to DTT had increased enzyme activity of complexes I (p = 0.009) and III (p = 0.018) of the electron transport system, while complexes II (p = 0.630) and IV (p = 0.926) showed no changes. However, increased enzyme activity did not carry over to measurements of mitochondrial respiration in permeabilized bundles. Oxidative phosphorylation conductance (p = 0.439), maximal respiration (p = 0.312), and ADP sensitivity (p = 0.514) were unchanged by 5 mM DTT treatment. These results indicate that mitochondrial function can be modulated through reversible thiol oxidation, but other components of mitochondrial energy transfer are rate limiting in end-stage HFrEF. Optimal therapies to normalize cardiac mitochondrial respiration in patients with end-stage HFrEF may benefit from interventions to reverse thiol oxidation, which limits complex I and III activities.

Vanadium pentoxide induced oxidative stress and cellular senescence in human lung fibroblasts

Both environmental exposure to vanadium pentoxide (V2O5, V+5 for its ionic counterparts) and fibroblast senescence are associated with pulmonary fibrosis, but whether V+5 causes fibroblast senescence remains unknown. We found in a dose-response study that 2–40 μM V+5 caused human lung fibroblasts (HLF) senescence with increased senescence-associated β-galactosidase activity and p16 expression, while cell death occurred at higher concentration (LC50, 82 μM V+5). Notably, measures of reactive oxygen species (ROS) production with fluorescence probes showed no association of ROS with V+5-dependent senescence. Preloading catalase (polyethylene-conjugated), a H2O2 scavenger, did not alleviate the cellular senescence induced by V+5. Analyses of the cellular glutathione (GSH) system showed that V+5 oxidized GSH, increased GSH biosynthesis, stimulated cellular GSH efflux and increased protein S-glutathionylation, and addition of N-acetyl cysteine inhibited V+5-elevated p16 expression, suggesting that thiol oxidation mediates V+5-caused senescence. Moreover, strong correlations between GSSG/GSH redox potential (Eh), protein S-glutathionylation, and cellular senescence (R2 > 0.99, p < 0.05) were present in V+5-treated cells. Studies with cell-free and enzyme-free solutions showed that V+5 directly oxidized GSH with formation of V+4 and GSSG in the absence of O2. Analyses of V+5 and V+4 in HLF and culture media showed that V+5 was reduced to V+4 in cells and that a stable V+4/V+5 ratio was rapidly achieved in extracellular media, indicating ongoing release of V+4 and reoxidation to V+5. Together, the results show that V+5-dependent fibroblast senescence is associated with a cellular/extracellular redox cycling mechanism involving the GSH system and occurring under conditions that do not cause cell death. These results establish a mechanism by which environmental vanadium from food, dietary supplements or drinking water, can cause or contribute to lung fibrosis in the absence of high-level occupational exposures and cytotoxic cell death.

From guide to guard—activation mechanism of the stress-sensing chaperone Get3

Oxidative stress conditions can cause ATP depletion, oxidative protein unfolding, and potentially toxic protein aggregation. To alleviate this proteotoxic stress, the highly conserved yeast protein, Get3, switches from its guiding function as an ATP-dependent targeting factor for tail-anchored proteins to its guarding function as an ATP-independent molecular chaperone that prevents irreversible protein aggregation. Here, we demonstrate that activation of Get3's chaperone function follows a tightly orchestrated multi-step process, centered around the redox status of two conserved cysteines, whose reactivity is directly controlled by Get3's nucleotide-binding state. Thiol oxidation causes local unfolding and the transition into chaperone-active oligomers. Vice versa, inactivation requires the reduction of Get3's cysteines followed by ATP-binding, which allows the transfer of bound client proteins to ATP-dependent chaperone systems for their effective refolding. Manipulating this fine-tuned cycle of activation and inactivation in yeast impairs oxidative stress resistance and growth, illustrating the necessity to tightly control Get3's intrinsic chaperone function.

Nonlinear Behavior during the Electrochemical Oxidation of Thiols

The electrochemical behavior of sulfur-containing compounds is of great importance in fields ranging from diagnostics to energy-storage and materials chemistry. For example, thiols (R-SH) are important components of many electrochemical sensors and are routinely used in materials applications as they are known to form well-ordered and reproducible monolayers (self-assembled monolayers, SAMs) on various metal surfaces. Sulfur compounds are also interesting from the viewpoint of nonlinear dynamics owing to the presence of multiple oxidation states, which has been exploited to study complex dynamic behavior in both chemical and electrochemical systems. In this work we explore the oscillatory behavior of 2-(dimethylamino)ethanethiol (DMAET), a known hydrolysis product of V-type nerve agents, on platinum electrodes. Interestingly, the electrochemical oxidation of DMAET leads to two distinct regions of oscillatory behavior. Probing the system with electrochemical impedance spectroscopy (EIS), resulted in the classification of the DMAET system as an HN-NDR type oscillator. These results are compared to structurally similar thiol containing compounds towards the goal of elucidating general features which permit the evolution of dynamic behavior in thiol-based electrochemical systems.

Silver Clusters of Five Atoms as Highly Selective Antitumoral Agents Through Irreversible Oxidation of Thiols (Adv. Funct. Mater. 29/2022)

Silver Clusters of Five Atoms as Highly Selective Antitumoral Agents Through Irreversible Oxidation of Thiols (Adv. Funct. Mater. 29/2022)

Structural snapshots of nitrosoglutathione binding and reactivity underlying S-nitrosylation of photosynthetic GAPDH

S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

The effect of hydrogen peroxide concentration and water temperature on yellowtail kingfish Seriola lalandi in a repeated bathing treatment

Hydrogen peroxide is routinely used in commercial yellowtail kingfish culture to treat monogenean infections. As the culture of this species expands into warmer geographical areas, this study aimed to compare the effects of a repeated exposure to different hydrogen peroxide concentrations at two rearing temperatures, 18 and 26 °C. The repeat bath occurred 14 days after the initial bath in line with commercial treatment regimes. The three concentrations tested were 85, 170 and 340 mg L−1, with bathing conducted for 30 min. Mortality occurred following the second bath at 340 mg L−1 at 26 °C.The highest hydrogen peroxide dose caused epithelial lifting in gill filaments and significantly reduced the size and abundance of common mucus cells within the skin epithelium following the repeat bath in warm water. Despite the highest dose causing gill damage and mortality in warm water, there were no significant effects of hydrogen peroxide concentration on any of the conventional blood parameters, except in moribund fish. A novel measurement of plasma thiol oxidation, however, was found to be significantly elevated at the highest bathing concentration.Whilst yellowtail kingfish appeared to be tolerant of the high concentration of hydrogen peroxide tested in the current study at ambient water temperature, these fish were not infected with monogeneans, and any such burden is likely to increase the susceptibility of the fish to the effects of hydrogen peroxide.

Cysteine Oxidation Promotes Dimerization/Oligomerization of Circadian Protein Period 2.

The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.