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Skin aging, characterized by hyperpigmentation, inflammation, wrinkles, and skin cancer, is influenced by intrinsic and extrinsic factors with synergistic effects. Autophagy maintains the homeostatic balance between the degradation, synthesis, and recycling of cellular proteins and organelles, and plays important roles in several cellular and biological processes, including aging. The compound 7-methylsulfinylheptyl isothiocyanate (7-MSI) is a sulfur-containing phytochemical produced by various plants, particularly cruciferous vegetables, with reported anti-inflammatory properties and a role in pathogen defense; however, its effects on skin whitening have not been studied in detail. The purpose of this study was to observe the effects of 7-MSI on skin whitening and autophagy in cultured murine melanoma (B16-F1) cells. Western blotting was used to evaluate the impact of 7-MSI on melanogenesis-, tyrosinase-, and autophagy-associated proteins. The levels of the melanogenesis-associated protein's microphthalmia-associated transcription factor (MITF) and tyrosinase and tyrosinase-related protein-1 were decreased by treatment with 7-MSI under melanogenesis induction. Melanin synthesis also decreased by approximately 63% after treatment with 7-MSI for 73 h, compared with that non-treated controls. In addition, autophagosome formation and the expression levels of the autophagy-related proteins mTOR, p-mTOR, Beclin-1, Atg12, and LC3 were higher in 7-MSI-treated B16-F1 cells than in non-treated cells. These results indicate that 7-MSI can inhibit melanin synthesis in B16-F1 cells by suppressing melanogenesis and autophagy activation and thus can potentially be used as a novel multifunctional cosmetic agent.
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BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia and most of AD patients suffer from vascular abnormalities and neuroinflammation. There is an urgent need to develop novel blood biomarkers capable of diagnosing Alzheimer's disease (AD) at very early stage. This study was performed to find out new accurate plasma diagnostic biomarkers for AD by investigating a direct relationship between plasma contact system and AD. METHODS: A total 101 of human CSF and plasma samples from normal and AD patients were analyzed. The contact factor activities in plasma were measured with the corresponding specific peptide substrates. RESULTS: The activities of contact factors (FXIIa, FXIa, plasma kallikrein) and FXa clearly increased and statistically correlated as AD progresses. We present here, for the first time, the FXIIa cut-off scores to as: > 26.3 U/ml for prodromal AD [area under the curve (AUC) = 0.783, p < 0.001] and > 27.2 U/ml for AD dementia (AUC = 0.906, p < 0.001). We also describe the cut-off scores from the ratios of CSF Aß1-42 versus the contact factors. Of these, the representative ratio cut-off scores of Aß1-42/FXIIa were to be: < 33.8 for prodromal AD (AUC = 0.965, p < 0.001) and < 27.44 for AD dementia (AUC = 1.0, p < 0.001). CONCLUSION: The activation of plasma contact system is closely associated with clinical stage of AD, and FXIIa activity as well as the cut-off scores of CSF Aß1-42/FXIIa can be used as novel accurate diagnostic AD biomarkers.
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Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H2DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy.
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Autofagia , Endorfinas/metabolismo , Precursores de Proteínas/metabolismo , Envejecimiento de la Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Línea Celular , Dermis/citología , Dermis/metabolismo , Dermis/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Procolágeno/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: There is a need to identify and develop novel thrombolytic agents that can directly digest fibrin clots from various biological resources. OBJECTIVE: To clone, express, purify, and characterize a recombinant protease named rvFMP capable of cleaving fibrinogen, fibrin polymer, and cross-linked fibrin in human plasma milieu and rat thrombosis model systems. RESULTS: We cloned a vFMP-encoding gene from the genomic DNA of V. furnissii KCCM41679 using polymerase chain reaction (PCR), expressed in Escherichia coli, and purified rvFMP (stands for recombinant vibrio furnissii metalloprotease). The proteolytic activity of purified rvFMP enzyme could be clearly inhibited by 1,10-phenanthroline and ethylene glycol tetraacetic acid, but not by diisopropyl fluorophosphate, suggesting that it can be a typical metalloprotease. rvFMP showed an effective proteolytic activity in cleaving cross-linked fibrins in human plasma milieu. Remarkably, rvFMP exhibited a clear thrombolytic activity in rat thrombosis models such as ferric chloride-exposed rat carotid artery and carrageenan-treated rat tail. However, rvFMP (1.5 mg/kg) evoked no internal bleeding and also showed no lethal effect in mice. The recombinant enzyme also showed no cytotoxicity and had an inability to induce tumour necrosis factor-α (TNF-α) in Raw264.7 cells. CONCLUSION: rvFMP can be a candidate enzyme capable of being developed as a novel direct-acting thrombolytic agent.
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Clonación Molecular , Fibrinolíticos , Péptido Hidrolasas , Trombosis , Animales , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Humanos , Ratones , Plasma , Ratas , Trombosis/tratamiento farmacológico , VibrioRESUMEN
BACKGROUND: As interest in skin beauty increases, the development of new skin whitening agents has attracted substantial attention; however, the action mechanism of the agents developed so far remains largely unknown. Tranexamic acid (TXA) is commonly being used to reduce melanin synthesis in patients with melasma and also used as a raw material for functional whitening cosmetics, although its action mechanism is poorly understood. Autophagy has been well known to be essential for tissue homeostasis, adaptation to starvation, and removal of dysfunctional organelles or pathogens. Recent studies have shown that autophagy regulators might have prominent roles in the initial formation stage of the melanosome, a lysosome-related organelle synthesizing melanin pigments. However, there is still no direct evidence showing a relationship between the activation of the autophagy system and the melanogenesis. OBJECTIVE: To investigate whether TXA can inhibit melanogenesis through the activation of autophagy in a melanoma cell line. METHODS: B16-F1 melanoma cells were treated with TXA and the levels of autophagy- and melanogenesis-related proteins were determined by Western blottings. The direct effect of TXA-mediated autophagy activation on melanin production was further evaluated by transfecting the cells with 60 pmols of small interfering RNAs (siRNAs)-targeting the mechanistic target of rapamycin (mTOR) and the autophagy-related protein 5 (Atg5). RESULTS: The results of Western blottings showed that TXA enhanced the production of autophagy-related proteins such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK)1/2, Beclin-1, Atg12, and light chain 3 (LC3) I-II, whereas it decreased the synthesis of the mTOR complex. Confocal microscopy clearly showed that TXA treatment resulted in the formation of autophagosomes in B16-F1 cells, as revealed by immunostaining with an anti-LC3 antibody. The production of melanogenesis-associated proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 and 2 (TRP1/2), were clearly downregulated by the treatments with TXA. These results suggest that TXA can mediate a decrease in melanin synthesis by alleviating the production of tyrosinase and TRP1/2, along with lowered MITF protein levels. Furthermore, after treatment with TXA, siRNAs- targeting to mTOR and Atg5 increased melanin synthesis by 20% and 40%, respectively, compared to that in non-transfected cells, in a dose-dependent manner. These results further confirmed that TXA can inhibit melanogenesis by activating the autophagy system. CONCLUSION: Collectively, the results demonstrate that TXA can reduce melanin synthesis in melanoma B16-F1 cells by activating the ERK signaling pathway and the autophagy system.