Mitochondrial reactive oxygen is critical for IL-12/IL-18-induced IFN-γ production by CD4+ T cells and is regulated by Fas/FasL signaling.

Mitochondrial activation and the production of mitochondrial reactive oxygen species (mROS) are crucial for CD4+ T cell responses and have a role in naïve cell signaling after TCR activation. However, little is known about mROS role in TCR-independent signaling and in recall responses. Here, we found that mROS are required for IL-12 plus IL-18-driven production of IFN-γ, an essential cytokine for inflammatory and autoimmune disease development. Compared to TCR stimulation, which induced similar levels of mROS in naïve and memory-like cells, IL-12/IL-18 showed faster and augmented mROS production in memory-like cells. mROS inhibition significantly downregulated IFN-γ and CD44 expression, suggesting a direct mROS effect on memory-like T cell function. The mechanism that promotes IFN-γ production after IL-12/IL-18 challenge depended on the effect of mROS on optimal activation of downstream signaling pathways, leading to STAT4 and NF-κB activation. To relate our findings to IFN-γ-driven lupus-like disease, we used Fas-deficient memory-like CD4+ T cells from lpr mice. Importantly, we found significantly increased IFN-γ and mROS production in lpr compared with parental cells. Treatment of WT cells with FasL significantly reduced mROS production and the activation of signaling events leading to IFN-γ. Moreover, Fas deficiency was associated with increased mitochondrial levels of cytochrome C and caspase-3 compared with WT memory-like cells. mROS inhibition significantly reduced the population of disease-associated lpr CD44hiCD62LloCD4+ T cells and their IFN-γ production. Overall, these findings uncovered a previously unidentified role of Fas/FasL interaction in regulating mROS production by memory-like T cells. This apoptosis-independent Fas activity might contribute to the accumulation of CD44hiCD62LloCD4+ T cells that produce increased IFN-γ levels in lpr mice. Overall, our findings pinpoint mROS as central regulators of TCR-independent signaling, and support mROS pharmacological targeting to control aberrant immune responses in autoimmune-like disease.

Mercury-Tolerant Ensifer medicae Strains Display High Mercuric Reductase Activity and a Protective Effect on Nitrogen Fixation in Medicago truncatula Nodules Under Mercury Stress.

Mercury (Hg) is extremely toxic for all living organisms. Hg-tolerant symbiotic rhizobia have the potential to increase legume tolerance, and to our knowledge, the mechanisms underlying Hg tolerance in rhizobia have not been investigated to date. Rhizobial strains of Ensifer medicae, Rhizobium leguminosarum bv. trifolii and Bradyrhizobium canariense previously isolated from severely Hg-contaminated soils showed different levels of Hg tolerance. The ability of the strains to reduce mercury Hg2+ to Hg0, a volatile and less toxic form of mercury, was assessed using a Hg volatilization assay. In general, tolerant strains displayed high mercuric reductase activity, which appeared to be inducible in some strains when grown at a sub-lethal HgCl2 concentration. A strong correlation between Hg tolerance and mercuric reductase activity was observed for E. medicae strains, whereas this was not the case for the B. canariense strains, suggesting that additional Hg tolerance mechanisms could be playing a role in B. canariense. Transcript abundance from merA, the gene that encodes mercuric reductase, was quantified in tolerant and sensitive E. medicae and R. leguminosarum strains. Tolerant strains presented higher merA expression than sensitive ones, and an increase in transcript abundance was observed for some strains when bacteria were grown in the presence of a sub-lethal HgCl2 concentration. These results suggest a regulation of mercuric reductase in rhizobia. Expression of merA genes and mercuric reductase activity were confirmed in Medicago truncatula nodules formed by a sensitive or a tolerant E. medicae strain. Transcript accumulation in nodules formed by the tolerant strain increased when Hg stress was applied, while a significant decrease in expression occurred upon stress application in nodules formed by the Hg-sensitive strain. The effect of Hg stress on nitrogen fixation was evaluated, and in our experimental conditions, nitrogenase activity was not affected in nodules formed by the tolerant strain, while a significant decrease in activity was observed in nodules elicited by the Hg-sensitive bacteria. Our results suggest that the combination of tolerant legumes with tolerant rhizobia constitutes a potentially powerful tool in the bioremediation of Hg-contaminated soils.

Exploration of Recombinant Streptokinase Degradation Products Under Various pH Reduction Conditions in Downstream Processing.

BACKGROUND: Methods of producing streptokinase, which can be used in the treatment of myocardial infarction, by hemolytic streptococci and recombinant E. coli have been described in patents since 1955. Degradation products in active pharmaceutical ingredients (APIs) and finished pharmaceutical products are considered as impurities and it is required that these degradation impurities are minimized or rather avoided throughout manufacturing process. OBJECTIVE: The aim of this study was to explore the occurrence of rSK degradation during acidification step in downstream processing. METHODS: The polyclonal antibody was produced by immunization of New Zealand white (NZW) rabbit with pure rSK (purity>98%). The solubilized inclusion bodies with various pH values (4.2, 5.0 and 6.0) were analyzed by Western blotting using rSK polyclonal antibody. RESULTS: Western blot analysis demonstrated the generation of rSK degradation products (with the molecular weight of about 27, 20 and 17 kDa) when the pH value of the solubilized inclusion bodies was reduced to 5.0 and 4.2, while no degradation of rSK observed at pH 6.0. CONCLUSION: This study demonstrates that the level of pH reduction in the solubilized inclusion bodies during downstream processing plays an important role in generating rSK degradation products, and substantial post-solubilization degradation of rSK occurs at pH lower than 6.0. Development of these degradation impurities, which cannot be eliminated by subsequent chromatographic purifications, can be exclusively avoided during acidification procedure by appropriate pH adjustment approach in downstream processing.

Effect of post-solubilization conditions on the yield and efficiency of recombinant streptokinase purification at large-scale.

Streptokinase, a plasminogen activator which converts plasminogen to plasmin and consequently promotes fibrinolysis, is the leading drug for treating acute myocardial infarction in developing countries and its production is industrially demanded. In this work, the substantial influence of inclusion body (IB) post-solubilization condition on the performance of a sequential chromatography method for large-scale purification of recombinant streptokinase was demonstrated. In the preliminary experiments, various post-solubilization pH conditions were studied, and it was shown that the pH value of solubilized inclusion bodies (i.e., in 4M urea) had a marked impact on the purity of streptokinase obtained at the end of post-solubilization process. When the pH value of the solution containing solubilized IBs was decreased from 7.5 to 6.5 and 6.0, the greatest increases (10% and 27%, respectively) in streptokinase purity occurred. The influence of different post-solubilization pH conditions on the efficiency and yield of large-scale chromatographic purification methods was next investigated. When the solubilized IBs solution with pH adjusted to 6.0 was utilized for subsequent sequential chromatography process, the complete elution peak with high overall yield (91.3%) and purity (98%) was achieved. In comparison to this, while the sequential chromatography procedure was instigated by using the solubilized IBs solution with pH 4.2, four elution fractions (EF1 to EF4) with disparate target protein purities (i.e., 57%, 77.3%, 91.4% and 86.7%, respectively) were attained, the process was incompletely effective, and the highest recovery and purity figures (81.8% and 91.4%, respectively, belonging to EF3) were much lower than those for the earlier process.