Hypobaria-Induced Oxidative Stress Facilitates Homocysteine Transsulfuration and Promotes Glutathione Oxidation in Rats with Mild Traumatic Brain Injury

Background: United States service members injured in combat theatre are often aeromedically evacuated within a few days to regional military hospitals. Animal and epidemiological research indicates that early exposure to flight hypobaria may worsen brain and other injuries. The mechanisms by which secondary exposure to hypobaria worsen trauma outcomes are not well elucidated. This study tested the hypothesis that hypobaria-induced oxidative stress and associated changes in homocysteine levels play a role in traumatic brain injury (TBI) pathological progression caused by hypobaria. Methods: Male Sprague Dawley rats were exposed to a 6 h hypobaria 24 h after mild TBI by the controlled cortical impact. Plasma and brain tissues were assessed for homocysteine levels, oxidative stress markers or glutathione metabolism, and behavioral deficits post-injury in the absence and presence of hypobaria exposure. Results: We found that hypobaria after TBI increased oxidative stress markers, altered homocysteine metabolism, and promoted glutathione oxidation. Increased glutathione metabolism was driven by differential upregulation of glutathione metabolizing genes. These changes correlated with increased anxiety-like behavior. Conclusion: These data provide evidence that hypobaria exposure after TBI increases oxidative stress and alters homocysteine elimination likely through enhanced glutathione metabolism. This pathway may represent a compensatory mechanism to attenuate free radical formation. Thus, hypobaria-induced enhancement of glutathione metabolism represents a potential therapeutic target for TBI management.

Forming active recombinant enterokinase expressed in Escherichia coli

Enterokinase is a serine protease commonly used in some biotechnology researches. For these purposes, the light chain containing enterokinase activity has usually been expressed as recombinant protein in different expression systems because natural enterokinase extraction is often ineffective. In this study, we examined the formation of recombinant enterokinase expressed in Escherichia coli with biological activity. The thioredoxin-enterokinase (trx-ent) fusion protein was autocleavaged into thioredoxin and enterokinase when expressed under insoluble form, denatured with guanidine and then refolded with suitable oxidation and reduction steps. Meanwhile, soluble expression as well as insoluble form denatured by urea had not enzymatic activity. Denaturant solution of 6 M guanidine along with the re-folding conditions in oxidized glutathione oxidation buffers followed by the reduced glutathione buffer with arginine was applied to produce trx-ent protein capable of self-cleavage. The recombinant light-chain enterokinase protein had a size of about 35 kDa on the Tris-glycine gel. Initial assessment on substance had shown that enterokinase was capable of cleaving thioredoxin-sumoprotease into thioredoxin and sumoprotease. This result provides the base for the production of active recombinant enterokinase to be used in recombinant protein expression technology.

Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells

One major challenge observed for the expression of therapeutic bispecific antibodies (BisAbs) is high product aggregates. Aggregates increase the risk of immune responses in patients and therefore must be removed at the expense of purification yields. BisAbs contain engineered disulfide bonds, which have been demonstrated to form product aggregates, if mispaired. However, the underlying intracellular mechanisms leading to product aggregate formation remain unknown. We demonstrate that impaired glutathione regulation underlies BisAb aggregation formation in a CHO cell process. Aggregate formation was evaluated for the same clonal CHO cell line producing a BisAb using fed-batch and perfusion processes. The perfusion process produced significantly lower BisAb aggregates compared to the fed-batch process. Perfusion bioreactors attenuated mitochondrial dysfunction and ER stress resulting in a favorable intracellular redox environment as indicated by improved reduced to oxidized glutathione ratio. Conversely, mitochondrial dysfunction-induced glutathione oxidation and ER stress disrupted the intracellular redox homeostasis, leading to product aggregation in the fed-batch process. Combined, our results demonstrate that mitochondrial dysfunction and ER stress impaired glutathione regulation leading to higher product aggregates in the fed-batch process. This is the first study to utilize perfusion bioreactors as a tool to demonstrate the intracellular mechanisms underlying product aggregation formation.

Immunotoxicity in Ascidians: Antifouling Compounds Alternative to Organotins—V. the Case of Dichlofluanid

Dichlofluanid has long been employed as a fungicide in agriculture and has been massively introduced in antifouling paints for boat hulls over the last two decades. One of the most important toxic effects of antifoulants is represented by immunosuppression in marine invertebrates, which can be analysed in vitro with a number of short-term toxicity assays on haemocytes. Among bioindicators, the colonial ascidian Botryllus schlosseri is a useful candidate; it is a filter-feeding organism living in the water-sediment interface that is found worldwide and is sensitive to antifouling xenobiotics. Dichlofluanid adversely affects both immunocyte lines (phagocyte and cytotoxic lines) after exposure to sublethal concentrations. At 0.05 μM (16.65 μg/L), dichlofluanid induced haemocyte apoptosis and cell shrinkage with a decrease in both motility and phagocytosis. At the lowest concentration (0.01 μM, 3.33 μg/L), inhibition of pivotal enzymatic activities of phagocytes and cytotoxic cells occurred. At the highest concentration (0.1 μM, 33.3 μg/L), dichlofluanid increased glutathione oxidation, leading to stress conditions. The effects of dichlofluanid on immune defence responses are similar to those of organometal-based antifoulants (i.e., organotin compounds and zinc pyrithione), and its use in coastal areas requires attention.

Protective Effects of Edaravone Against Hypoxia-Induced Lethality in Mice

Edaravone is used for the treatment of acute cerebral infarction in Japan. However, nothing is known about the protective effects of this drug against hypoxia-induced lethality. In this study, the protective effects of edaravone against hypoxia-induced lethality and oxidative stress in mice were evaluated by three experimental models of hypoxia, which are asphyctic, haemic, and circulatory. Statistically significant protective activities were established in all tested models of hypoxia. Antihypoxic activities were especially pronounced in asphytic and circulatory hypoxia. The effect was dose-dependent. Edaravone, at 5 mg kg-1, showed statistically significant activities respect to the control groups. It significantly prolonged the latency for death. At 2.5 mg kg-1, it also prolonged survival time (26.08 ± 5.26 min), but this effect was not statistically significant from the control (P>0.05). On the other hand, edaravone significantly inhibited hypoxia-induced oxidative stress (lipid peroxidation and glutathione oxidation) in three models of hypoxia. In conclusion, the results obtained in this study showed that Edaravone has very good protective effects against the hypoxia in all tested models.

A Melanin-Related Phenolic Polymer with Potent Photoprotective and Antioxidant Activities for Dermo-Cosmetic Applications

Eumelanins, the dark variant of skin pigments, are endowed with a remarkable antioxidant activity and well-recognized photoprotective properties that have been ascribed to pigment components derived from the biosynthetic precursor 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Herein, we report the protective effect of a polymer obtained starting from the methyl ester of DHICA (MeDHICA-melanin) against Ultraviolet A (UVA)-induced oxidative stress in immortalized human keratinocytes (HaCaT). MeDHICA-melanin was prepared by aerial oxidation of MeDHICA. At concentrations as low as 10 µg/mL, MeDHICA-melanin prevented reactive oxygen species accumulation and partially reduced glutathione oxidation in UVA-irradiated keratinocytes. Western blot experiments revealed that the polymer is able to induce the translocation of nuclear factor erythroid 2–related factor 2 (Nrf-2) to the nucleus with the activation of the transcription of antioxidant enzymes, such as heme-oxygenase 1. Spectrophotometric and HPLC analysis of cell lysate allowed to conclude that a significant fraction (ca. 7%), consisting mainly of the 4,4′-dimer of MeDHICA (ca. 2 μM), was internalized in the cells. Overall these data point to the potential use of MeDHICA-melanin as an antioxidant for the treatment of skin damage, photoaging and skin cancers.

NADPH and glutathione redox link TCA cycle activity to endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is associated with dysregulated metabolism, but little is known about how the ER responds to metabolic activity. Here, working primarily in mouse hepatocytes, we show that decreasing the availability of substrate for the TCA cycle diminished NADPH production and attenuated ER stress in a manner that depended on glutathione oxidation. ER stress was also alleviated by impairing either TCA-dependent NADPH production or Glutathione Reductase. Conversely, stimulating TCA activity favored NADPH production, glutathione reduction, and ER stress. Validating these findings, we show that deletion of the mitochondrial pyruvate carrier, which is known to decrease TCA cycle activity and protect the liver from diet-induced injury, also diminished NADPH, elevated glutathione oxidation, and alleviated ER stress. These results provide independent genetic evidence that mitochondrial oxidative metabolism is linked to ER homeostasis. Our results demonstrate a novel pathway of communication between mitochondria and the ER, through relay of redox metabolites.