<p>Hydrogen peroxide (H₂O₂), a reactive oxygen specie (ROS), is most commonly associated with oxidative stress causing cytotoxic effects on living cells. Oxidative stress has been implicated in various conditions including neurodegenerative diseases, autoimmune diseases and cancer. In addition H₂O₂is produced as a defense mechanism against pathogens, as being released by activated phagocytes.<em> </em>In recent years, H₂O₂ has become established as an important regulator of signal transduction in eukaryotic cells. Hydrogen peroxide is generated both intracellularly and extracellularly in response to various stimuli including cytokines and growth factors. There are different mechanisms by which H₂O₂ is generated, facilitating signal transduction in cells; through NOX-system in miyochondria, via singlet oxygen, receptor/ligand interaction or by redox active metal ions. The HIV glycoprotein 120 (gp120) is associated with HIV dementia and it is known as a neurotoxin that causes neuronal damage. It has been proposed that free radicals may be involved in the pathogenesis caused by gp120. In addition the truncated form of thioredoxin (Trx80) is known to stimulate HIV replication in HIV infected cells, however, the exact mechanism is not known. A possible way both proteins may mediate their activity is by inducing H₂O₂ production. The aim of this study was to investigate H₂O₂ production induced by the proteins gp120 and Trx80. In order to detect H₂O₂ production an assay based on the fluorescent compound Amplex Red, was established. The assay was used to detect H₂O₂ released by gp120 and Trx80 in a cell-free environment, in a cell-system and in the presence of metal ions (copper ions) with a physiological reductant (ascorbate). We did not detect H₂O₂ production induced by gp120 and Trx80 respectively, using our assay, however, other ROS such as hydroxyl radicals may have been generated although they were not detectable with our method. Hence, further studies are needed in order to fully understand how gp120 and Trx80 mediate their activity.</p>

This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite’s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™ Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.

Changes in intracellular redox homeostasis are implicated in both normal cell signaling and as pathophysiological mechanisms contributing to a variety of age-related diseases, including diabetes, atherosclerosis, neurodegenerative conditions, and cancer. Though a variety of well described mechanisms exist to counterbalance the overproduction of cellular oxidants and maintain optimal intracellular redox poise, the understanding of the mechanism(s) through which cellular redox homeostasis regulates cell signaling functions is less well understood. Here, we demonstrate that signaling by the immediate early gene / orphan nuclear hormone receptor NGFI-B (Nur77, TR3), which functions pleiotropically in the regulation of cell growth, metabolism, differentiation and death in diverse tissues, is redox-regulated at both the level of induction and NGFI-B-dependent gene transcription. Using co-immunoprecipitation experiments in cells, we also identified a novel interaction between NGFI-B and the cytoplasmic thiol-reducing catalyst thioredoxin1 (Trx1), that, similar to DTT, blocks NGFI-B-dependent gene expression in a manner that depends on the Trx1 active site cysteines. Together these observations add NGFI-B-dependent gene expression to a growing portfolio of transcription factor pathways that are redox-regulated. NGFI-B, in addition, appears to regulate the mitochondrial membrane potential in L6 skeletal myoblasts. NGFI-B is indispensible for T-cell receptor-mediated apoptosis and induces cell death in a variety of cell types in response to diverse pro-apoptotic stimuli. Like p53, translocation of NGFI-B from the nucleus to the mitochondria may be a critical aspect of its pro-apoptotic function. Interestingly, we found that enforced NGFI-B expression in L6 skeletal muscle myoblasts led to a significant decrease of MMP that peaked 48hr after transfection and did not require a cell death-inducing stimulus. Moreover, NGFI-B transfected cells had no increase in mitochondrial cytochrome C release despite loss of MMP at 48 hr. Combined, these data suggest that loss of MMP in muscle cells may be an early event in the apoptotic process regulated by NGFI-B. This, along with the redox regulation of NGFI-B, provides unique evidence of a relationship between the mitochondria, mitochondrial by-products, ROS, and the regulation of and by the transcription factor NGFI-B. / text

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