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Trichoderma reesei superoxide dismutase (TrSOD) is a well-characterized enzyme being stable between 30 and 90 • C for 1 h with activity at pH between 2.6 and 9.0. This work aimed to clone, express, purify, and evaluate the protective effect antioxidant of this enzyme on skin cells when fused to transactivator of transcription (TAT) protein transduction domain of HIV-1 and abalone (Ab) peptides to allow cell penetration. TrSOD, TAT-TrSOD-Yfp (fused to yellow fluorescent protein), and Ab-TrSOD were expressed in E. coli and purified as soluble proteins. The cytotoxicity of the enzymes, at the concentrations of 1, 3, and 6 μmol/L, was evaluated for a period of 24 and 48 h of incubation, with no cytotoxic effect on 3T3 fibroblasts. The 3T3 cells were exposed to the oxidant agent tert-butyl hydroperoxide and evaluated for reactive oxygen species (ROS) generation, in the presence or not of the recombinant enzymes. TAT-TrSOD-Yfp was able to decrease the generation of ROS by 15% when used in the concentrations of 3 and 6 μmol/L in comparison to the control, but there was no difference in relation to the effect of TrSOD. Ab-TrSOD, when compared to TrSOD, promoted a decrease in the formation of ROS of 19% and 14% at the concentrations of 1 and 6 μmol/L, respectively, indicating that this recombinant form was more effective in reducing oxidative stress compared to SOD without the cell-penetrating peptide (CPP). Together, these results indicate that the fusion of SOD with these CPP increased the antioxidant capacity of fibroblasts, identified by the reduction in the generation of ROS. In addition, such molecules, in the concentrations initially used, were not toxic to the cells, opening perspectives for the development

Introduction: Type 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. The use of herbal medicines as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the Ut aqueous extract effect on cell viability of skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA), and on the reactive oxygen species (ROS) production, which consists a central event involved in T2DM pathogenesis. Methods: Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), at 37°C humidified atmosphere and 5% CO2. The cells were incubated with PA in different concentrations, in the presence or absence of 250 μg/ml Ut aqueous extract, for 2, 6 or 24 h. After these periods, oxidative stress was evaluated by fluorescence spectroscopy. Results: Incubation of cells with PA and Ut aqueous extract resulted in an increase of, at least, 50% in cell viability compared to control with only PA. The treatment of cells with Ut aqueous extract, for 6 h, followed by exposure to 500 μM PA, caused 38% less ROS formation than those incubated with only the free fatty acid. Conclusion: The Ut aqueous extract promoted a rise in cell viability, reduced cell death and attenuated ROS formation in cultures incubated with 500 μM PA, protecting cells from the fatty acid lipotoxicity.

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Different approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSC) from umbilical cord can be safely and easily obtained, however a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte terminal differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under control of pINV to assess MSC differentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, originating LeGO-GpINV. MSC were transduced with the LeGO-GpINV and induced to differentiate into keratinocytes upon cultivation with Keratinocyte Serum Free Medium supplemented. MSC differentiation was confirmed by morphological changes and by the expression of epidermal markers, by flow cytometry, quantitative PCR and western blot. The activity of kallikreins 5, 6 and 7 was detected using fluorogenic substrates. After 14 days of differentiation, MSC transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in the kallikrein activity. This reporter system allowed to temporally assess the epidermal differentiation, simultaneously with involucrin expression, opening perspectives for the in vivo study of skin biology and in regenerative medicine.