Gestational diabetes (GD) is a condition defined as carbohydrate intolerance and hyperglycemia beginning in the second trimester of pregnancy, which overlaps with the progesterone exponential increase. Progesterone has been shown to cause pancreatic β-cell death by a mechanism dependent on the generation of reactive oxygen species and oxidative stress. Herein, we studied the effect of this hormone on the expression of 84 genes related to oxidative stress and oxidant defense in pancreatic RINm5F cell lineage. Cells were incubated with 0.1, 1.0, or 100 μM progesterone for 6 or 24 h, in the presence or absence of the vitamins E and C. Among the investigated genes, five of them had their expression increased, at least 2-fold, in two different concentrations independently of the time of incubation, or at the same concentration at the different time points, including those that encode for stearoyl-CoA desaturase 1 (Scd1), dual oxidase 1 (Duox1), glutathione peroxidase 6 (GPx6), heme oxygenase 1 (Hmox1), and heat shock protein a1a (Hspa1a). Vitamins E and C were able to increase, in progesterone-treated cells, the expression of genes with antioxidant function such as Hmox1, but decreased Scd1 expression, a gene with prooxidant function. At cytoplasmic level, progesterone positively modulated Hmox1 and Hspa1a content. These results suggest that the protein encoded by these genes might protect cells against progesterone induced-oxidative damage, opening perspectives to elucidate the molecular mechanism involved in progesterone action in GD, as well as for the development of antioxidant strategies for the prevention and treatment of this disease.

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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

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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.