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  • In general cellular senescence is considered a programmed re


    In general, cellular senescence is considered a programmed response to stress that can be activated by oxidative stress, irradiation or action of substances or drugs. These stressors cause DNA damage and, through the action of specific proteins (p53 and p21), lead to cell senescence (di Fagagna, 2008). Assays that evaluate DNA damage as a cause of senescence are usually carried out in a short time, within 24h, considering that in longer periods repair mechanisms can be activated. For that reason, this experiment was performed at 6 and 12h, differing from other treatment times (48h) used in the present study. Thus, DNA damage appears to be a key aspect that would justify the presence of senescence in cells supplemented with Leu. The potential toxic effect of Leu and its relation to DNA damage has mainly been studied in pathological situations in which this amino Clotrimazole synthesis is not metabolized properly and ends up accumulating in the body, as in the case of patients with maple syrup urine disease (MSUD) (Strauss et al., 2013). Studies in vitro under conditions that mimic toxic environment by excessive Leu, as well as in individuals with MSUD, show that excess of Leu may cause cellular damage increased by DNA rupture (Mescka et al., 2015). This damage can occur even in cells from healthy individuals (Mescka et al., 2014). Our results indicate that Leu also has a potential toxic effect on MC3T3-E1 cells, since its 12.5% increase, as compared to the levels that are considered optimal for the culture medium of these cells, was able to promote DNA damage and, consequently, cellular senescence at 6 and 12h of treatment.
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    Introduction The ubiquitin-proteasome system is a eukaryotic mechanism of intracellular protein degradation, which regulates basic cellular processes such as cell cycle, division, differentiation, and death [[1], [2], [3]]. Therefore, aberrations of this system are closely related to the pathogenesis of human diseases. Ubiquitination is catalysed by the sequential action of a single ubiquitin-activating enzyme E1 (UBE1) and multiple ubiquitin-conjugating E2 (UBE2) and ubiquitin-protein ligase E3 (UBE3) enzymes, and ubiquitinated proteins are finally degraded by proteasomes [3]. Since UBE1 is the common gatekeeping enzyme for ubiquitination, inhibition of UBE1 enzymatic activity blocks the ubiquitin-proteasome system. Target proteins are degraded by the 26S proteasome after ligation with lysine 48 (K48)-linked ubiquitin chains [3]. Otherwise, K63-linked ubiquitin chains contribute to the cytokine-induced activation of nuclear factor-κB (NF-κB) [4]. The 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (hereafter PYR-41) and related pyrazones are cell-permeable UBE1 inhibitors identified in high-throughput screening of the UBE1–UBE2–UBE3 cascade, which selectively and irreversibly inhibits UBE1 activity [3]. PYR-41 and other related pyrazones enter cells and decrease UBE1-ubiquitin thiolester formation via covalent modification, resulting in irreversible inhibition of the enzymatic activity of UBE1 [[5], [6]]. Since PYR-41 exposure inhibits NF-κB activation and increases the expression level and activity of p53, an anti-tumour protein, it is considered a potent drug for selectively killing transformed cells [5]. Dysfunction of the ubiquitin-proteasome system causes abnormal organelle morphology and impairs sperm formation and maturation steps resulting in male infertility [[7], [8], [9]]; this indicates that ubiquitination is essential for spermatogenesis and sperm function during fertilization. β-catenin is a well-known target protein for ubiquitination, and its cellular level is tightly controlled by ubiquitination-mediated degradation [10]. In fertilization, β-catenin degradation is triggered by membrane adhesion between sperm and egg, and treatment of wild-type eggs, but not β-catenin-deficient eggs, with PYR-41 affects sperm-egg fusion [11]. To further investigate the effect of PYR-41 on egg function, we here examined wild-type and β-catenin-deficient eggs pretreated or treated continuously with PYR-41.