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Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative, and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties, and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to nontarget control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.
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Glutatión Transferasa , Melaninas , Melanoma , Animales , Humanos , Ratones , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Melaninas/biosíntesis , Melanoma/genética , Melanoma/inmunología , Melanoma/fisiopatología , Pez Cebra/metabolismo , Oxidación-Reducción , Ratones Endogámicos C57BL , Línea Celular Tumoral , Proliferación Celular/genéticaRESUMEN
BACKGROUND: It is well documented that smokers suffer increased risk of postoperative complications after medical surgery, for example delayed healing and increased risk of infection. It is also known that preoperative smoking cessation can reduce the risk of these complications. Because of this there are guidelines regarding preoperative smoking cessation in non-oral medical surgery. There are however no specific guidelines regarding oral surgical procedures, such as surgical extractions, dentoalveolar surgery, periodontal surgery, or dental implantation. Nevertheless, it is common that dentists and oral surgeons recommend smoking cessation pre to oral surgical procedures. The aim with this systematic review was to see if there are any evidence in the literature, supporting preoperative smoking cessation in oral surgical procedures. METHODS: A systematic search of the electronic databases PubMed, Scopus, Web of Science, and Cochrane was conducted to identify studies addressing the effect of preoperative smoking cessation in oral surgical procedures. Included publications were subjected to preidentified inclusion criterion. Six examiners performed the eligibility and quality assessment of relevant studies. Risk of bias was assessed using ROBINS-I and RoB 2. Certainty assessment was carried out using GRADE. RESULTS: The initial search resulted in 2255 records, and after removal of 148 duplicates, 16 articles met an acceptable level of relevance. These were read in full text, whereof 12 articles were excluded, due to different intervention, outcome, or study design than stated in the review protocol. One study remained with moderate risk of bias and three were excluded due to high risk of bias. CONCLUSION: This systematic review could not determine the effect of smoking cessation pre to oral surgical procedures, in smokers. This indicates lack of knowledge in the effects of smoking cessation. We also conclude a lack of knowledge in how to design smoking cessation in the most effective way.
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Procedimientos Quirúrgicos Orales , Cese del Hábito de Fumar , Cicatrización de Heridas , Humanos , Procedimientos Quirúrgicos Orales/efectos adversos , Complicaciones Posoperatorias/prevención & control , Complicaciones Posoperatorias/etiología , FumadoresRESUMEN
While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.
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Antineoplásicos , Neoplasias Pulmonares , Melanoma , Humanos , Ratones , Animales , Glutatión Transferasa/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Estrés Oxidativo , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Glutatión/metabolismoRESUMEN
Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood. In this study, the impact of O6-methylguanine on the transcription fidelity of p53 and the subsequent effects on the protein's function as a regulator of cell death and cell-cycle arrest were examined in human cells. Levels of TM were determined by RNA-sequencing. In cells with active DNA repair, misincorporation of uridine opposite the lesion occurred in 0.14% of the transcripts and increased to 14.7% when repair by alkylguanine-DNA alkyltransferase was compromised. Expression of the dominant-negative p53 R248W mutant due to TM significantly reduced the transactivation of several established p53 target genes that mediate the tumor-suppressor function, including CDKN1A (p21) and BBC3 (PUMA). This resulted in deregulated signaling through the retinoblastoma protein and loss of G1/S cell-cycle checkpoint function. In addition, we observed impaired activation of apoptosis coupled to the reduction of the tumor-suppressor functions of p53. Taking these findings together, this work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for the role of TM in tumorigenesis.
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Transformación Celular Neoplásica/metabolismo , Guanina/análogos & derivados , Mutagénesis , Mutación Missense , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Sustitución de Aminoácidos , Apoptosis/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Reparación del ADN , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Guanina/metabolismo , Humanos , Puntos de Control de la Fase S del Ciclo Celular/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Caspase-2 has been implicated in diverse cellular processes, and the identification of factors with which it interacts has steadily increased. In the present study, we report a direct interaction between caspase-2 and factor associated with neutral sphingomyelinase activation (FAN) using yeast two-hybrid screening and co-immunoprecipitation. Further, stable suppression of caspase-2 expression in HEK293T and HeLa cells enabled a systematic investigation of putative novel enzyme functionalities, especially with respect to ceramide production, cell migration, IL-6 production and vesicular homeostasis, all of which have been previously reported to be associated with FAN. Lipidomics excluded the involvement of caspase-2 in the generation of ceramide species, but caspase-2-dependent deregulation of IL-6 release, vesicular size and delayed cell relocation supported an association between caspase-2 and FAN. Collectively, these data identify a novel caspase-2-interacting factor, FAN, and expand the role for the enzyme in seemingly non-apoptotic cellular mechanisms.
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Caspasa 2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Caspasa 2/deficiencia , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Ceramidas/farmacología , Células HEK293 , Humanos , Interleucina-6/metabolismo , Unión Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismoRESUMEN
The intercellular crosstalk between hematological malignancies and the tumor microenvironment is mediated by cell-to-cell interactions and soluble factors. One component of the secretome that is gaining increasing attention is the extracellular vesicles and, in particular, the exosomes. Apart from the role as vectors of molecular information, exosomes have been shown to possess intrinsic biological activity. In this study, we found that caspase-3 is activated in L88 bone marrow stroma cell-derived exosomes and identified 1 of the substrates to be the antiapoptotic protein Bcl-xL. The cleaved Bcl-xL is found in a panel of normal and cancer cell-derived exosomes and is localized on the outer leaflet of the exosomal membrane. Incubation of the exosomes with a caspase-3 inhibitor or the pan-caspase inhibitor prevents the cleavage of Bcl-xL. Importantly, MCF-7 cell-derived exosomes that are caspase-3-deficient are enriched in full-length Bcl-xL, whereas ectopic expression of caspase-3 restores the cleavage of Bcl-xL. Chemical inhibition of Bcl-xL with ABT737 or molecular inhibition by using the D61A and D76A Bcl-xL mutant leads to a significant decrease in the uptake of exosomes by hematopoietic malignant cells. These data indicate that the cleaved Bcl-xL is required for the uptake of exosomes by myeloma and lymphoma cells, leading to their increased proliferation. In summary, we demonstrate for the first time that Bcl-xL is an exosomal caspase-3 substrate and that this processing is required for the uptake of exosomes by recipient cells.
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Caspasa 3/metabolismo , Exosomas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Linfoma/metabolismo , Mieloma Múltiple/metabolismo , Células Madre Neoplásicas/metabolismo , Proteína bcl-X/metabolismo , Sustitución de Aminoácidos , Caspasa 3/genética , Exosomas/genética , Exosomas/patología , Femenino , Humanos , Células Jurkat , Linfoma/genética , Linfoma/patología , Células MCF-7 , Masculino , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Mutación Missense , Células del Estroma/metabolismo , Células del Estroma/patología , Proteína bcl-X/genéticaRESUMEN
Apoptosis signaling crucially depends on caspase activities. Caspase-2 shares features of both initiator and effector caspases. Opinions are divided on whether caspase-2 activity is established during apoptosis initiation or execution in response to DNA damage, death receptor stimulation, or heat shock. So far, approaches towards measuring caspase-2 activity were restricted to analyses in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. Furthermore, caspases overlap in substrate specificities, and caspase-8 as well as effector caspases may cleave the optimal VDVAD recognition motif as well. We therefore generated a highly sensitive Förster resonance energy transfer (FRET) substrate to determine the relative contribution of these caspases to VDVADase activity non-invasively inside living cells. We observed limited proteolysis of the substrate during apoptosis initiation in response to death receptor stimulation by FasL, TNFα and TRAIL. However, this activity was attributable to caspase-8 rather than caspase-2. Likewise, no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), microtubule destabilization (vincristine), or heat shock. The contribution of caspase-2 to proteolytic activities during apoptosis execution was insignificant. Since even residual, ectopically introduced caspase-2 activity could readily be detected inside living cells in our measurements, we conclude, in contrast to several previous studies, that caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signaling.
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Apoptosis/efectos de los fármacos , Caspasa 2/metabolismo , Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Caspasas Efectoras/metabolismo , Mitocondrias/efectos de los fármacos , Animales , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 2/química , Caspasa 8/química , Caspasas Efectoras/química , Células Cultivadas , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Calor , Humanos , Ratones , Mitocondrias/metabolismo , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current treatments have increased overall survival but can lead to devastating side effects and late complications in survivors, emphasizing the need for new, improved targeted therapies that specifically eliminate tumor cells while sparing the normally developing brain. METHODS: Here, we used a SHH-MB model based on a patient-derived neuroepithelial stem (NES) cell system for an unbiased high-throughput screen with a library of 172 compounds with known targets. Compounds were evaluated in both healthy neural stem cells and tumor cells derived from the same patient. Based on the difference of cell viability and drug sensitivity score between normal cells and tumor cells, hit compounds were selected and further validated in vitro and in vivo. RESULTS: We identified PF4708671 (S6K1 inhibitor) as a potential agent that selectively targets Sonic Hedgehog (SHH) driven MB tumor cells while sparing neural stem cells and differentiated neurons. Subsequent validation studies confirmed that PF4708671 inhibited the growth of SHH-MB tumor cells both in vitro and in vivo, and that knockdown of S6K1 resulted in reduced tumor formation. CONCLUSIONS: Overall, our results suggest that inhibition of S6K1 specifically affects tumor growth, whereas it has less effect on non-tumor cells. Our data also show that the NES cell platform can be used to identify potentially effective new therapies and targets for SHH-MB.
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Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.
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Compuestos de Boro , Poliuretanos , Humanos , Poliuretanos/toxicidad , Poliuretanos/química , Compuestos de Boro/química , Compuestos de Boro/toxicidad , Línea Celular , Piel/efectos de los fármacos , Piel/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Citocinas/metabolismo , Supervivencia Celular/efectos de los fármacosRESUMEN
The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg®) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.
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OBJECTIVE: The aim of this study was to evaluate fetal hemoglobin (HbF) and α(1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE). STUDY DESIGN: The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis. RESULTS: The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate. CONCLUSION: The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE.
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alfa-Globulinas/análisis , Sangre Fetal/química , Hemoglobina Fetal/análisis , Preeclampsia/sangre , Preeclampsia/diagnóstico , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Valor Predictivo de las Pruebas , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del EmbarazoRESUMEN
Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.
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Copper oxide nanoparticles (CuO NPs) have previously been shown to cause dose-dependent pulmonary toxicity following inhalation. Here, CuO NPs (10 nm), coated with polyethylenimine (PEI) or ascorbate (ASC) resulting in positively or negatively charged NPs, respectively, were evaluated. Rats were exposed nose-only to similar exposure dose levels of ASC or PEI coated CuO NPs for 5 consecutive days. On day 6 and day 27 post-exposure, pulmonary toxicity markers in bronchoalveolar lavage fluid (BALF), lung histopathology and genome-wide transcriptomic changes in lungs, were assessed. BALF analyses showed a dose-dependent pulmonary inflammation and cell damage, which was supported by the lung histopathological findings of hypertrophy/hyperplasia of bronchiolar and alveolar epithelium, interstitial and alveolar inflammation, and paracortical histiocytosis in mediastinal lymph nodes for both types of CuO NPs. Transcriptomics analysis showed that pathways related to inflammation and cell proliferation were significantly activated. Additionally, we found evidence for the dysregulation of drug metabolism-related genes, especially in rats exposed to ASC-coated CuO NPs. Overall, no differences in the type of toxic effects and potency between the two surface coatings could be established, except with respect to the (regional) dose that initiates bronchiolar and alveolar hypertrophy. This disproves our hypothesis that differences in surface coatings affect the pulmonary toxicity of CuO NPs.
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Enfermedades Pulmonares , Nanopartículas , Animales , Cobre/toxicidad , Hipertrofia , Inflamación , Exposición por Inhalación/efectos adversos , Nanopartículas/toxicidad , Óxidos , Ratas , TranscriptomaRESUMEN
UNLABELLED: The diagnostic medical image contains, apart from the pixel data, a detailed description of how the image was produced. The information reveals details on image geometry, radiation data as well as more complex quality index in a varying degree, mostly dependent on the age of the equipment. There is no simple way to retrieve, process and display this data in a general image workstation. MATERIAL AND METHODS: Since November 2004 a DICOM metadata repository has been used to record image header parameters. The automated data extraction, storage and display are based on simple standard programming and have performed without malfunction since the start, today containing metadata from 18 million images. RESULTS: The data in the metadata repository has been used in dose optimization for a Computed Radiography image plate system, analyzing the exposure index and making use of the possibilities to organize the data in examinations, projections as well as examination rooms. Analysis of exposure index in the context of these parameters shows promising qualities as it makes detection of dosimetric problems as well as follow-up of dose adjustments simpler. Current work is aimed at creating a vendor independent platform and to further develop methods to support dose optimization for flat panel direct digital detectors and computed tomography (CT) systems. The possibilities to detect equipment malfunction will be further investigated.
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Bibliotecas Digitales , Intensificación de Imagen Radiográfica , Sistemas de Información Radiológica , Tecnología Radiológica , Algoritmos , Calibración , Bases de Datos Factuales , Humanos , Modelos Biológicos , Protección Radiológica , Intensificación de Imagen Radiográfica/métodos , Dosificación Radioterapéutica , Tecnología Radiológica/métodos , Tomógrafos Computarizados por Rayos XRESUMEN
Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2-/- mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.
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Caspasa 2/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Sanguíneas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas de Unión al ADN , Doxorrubicina/farmacología , Fluorouracilo/farmacología , Células HCT116 , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Unión Proteica , Proteolisis , Reproducibilidad de los Resultados , Saccharomyces cerevisiae , Técnicas del Sistema de Dos HíbridosRESUMEN
The rapid dissolution of copper oxide (CuO) nanoparticles (NPs) with release of ions is thought to be one of the main factors modulating their toxicity. Here we assessed the cytotoxicity of a panel of CuO NPs (12â¯nm⯱â¯4â¯nm) with different surface modifications, i.e., anionic sodium citrate (CIT) and sodium ascorbate (ASC), neutral polyvinylpyrrolidone (PVP), and cationic polyethylenimine (PEI), versus the pristine (uncoated) NPs, using a murine macrophage cell line (RAW264.7). Cytotoxicity, reactive oxygen species (ROS) production, and cellular uptake were assessed. The cytotoxicity results were analyzed by the benchmark dose (BMD) method and the NPs were ranked based on BMD20 values. The PEI-coated NPs were found to be the most cytotoxic. Despite the different properties of the coating agents, NP dissolution in cell medium was only marginally affected by surface modification. Furthermore, CuCl2 (used as an ion control) elicited significantly less cytotoxicity when compared to the CuO NPs. We also observed that the antioxidant, N-acetylcysteine, failed to protect against the cytotoxicity of the uncoated CuO NPs. Indeed, the toxicity of the surface-modified CuO NPs was not directly linked to particle dissolution and subsequent Cu burden in cells, nor to cellular ROS production, although CuO-ASC NPs, which were found to be the least cytotoxic, yielded lower levels of ROS in comparison to pristine NPs. Hierarchical cluster analysis suggested, instead, that the toxicity in the current in vitro model could be explained by synergistic interactions between the NPs, their dissolution, and the toxicity of the coating agents.
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Muerte Celular/efectos de los fármacos , Cobre/toxicidad , Macrófagos/metabolismo , Nanopartículas del Metal/toxicidad , Animales , Antioxidantes , Línea Celular , Cobre/química , Cobre/farmacocinética , Nanopartículas del Metal/química , Ratones , Especies Reactivas de Oxígeno/metabolismo , Solubilidad , Propiedades de SuperficieRESUMEN
alpha(1)-Microglobulin is a 26-kDa glycoprotein synthesized in the liver, secreted to the blood, and rapidly distributed to the extravascular compartment of all tissues. Recent results show that alpha(1)-microglobulin has heme-binding and heme-degrading properties and it has been suggested that the protein is involved in the defense against oxidation by heme and reactive oxygen species. In the present study the influence of hemoglobin and reactive oxygen species (ROS) on the cellular expression of alpha(1)-microglobulin was investigated. Oxy- and methemoglobin, free heme, and Fenton reaction-induced hydroxyl radicals induced a dose-dependent up-regulation of alpha(1)-microglobulin on both mRNA and protein levels in hepatoma cells and an increased secretion of alpha(1)-microglobulin. The up-regulation was reversed by the addition of catalase and ascorbate, and by reacting hemoglobin with cyanide which prevents redox reactions. Furthermore, the blood cell lines U937 and K562 expressed alpha(1)-microglobulin at low levels, and this expression increased up to 11-fold by the addition of hemoglobin. These results suggest that alpha(1)-microglobulin expression is induced by ROS, arising from redox reactions of hemoglobin or from other sources and are consistent with the hypothesis that alpha(1)-microglobulin participates in the defense against oxidation by hemoglobin, heme, and reactive oxygen species.
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alfa-Globulinas/biosíntesis , Carcinoma Hepatocelular/metabolismo , Regulación de la Expresión Génica , Hemoglobinas/metabolismo , Especies Reactivas de Oxígeno , Regulación hacia Arriba , Línea Celular , Eritrocitos/metabolismo , Hemo/química , Humanos , Células K562 , Oxidación-Reducción , Oxígeno/metabolismo , ARN Mensajero/metabolismo , Células U937RESUMEN
BACKGROUND: Digital radiography has several advantages over screen-film radiography in data storage and retrieval, making it an attractive alternative to screen-film radiography in screening mammography programs, if it performs as well. METHODS: We retrospectively compared screen-film mammography, photon-counting direct radiography, and computed radiography with population-based screening data from the Breast Unit at Helsingborg Hospital, Sweden, collected between January 2000 and February 2005. Outcomes were cancer detection rates, recall rates, and positive predictive values for breast cancer detection in women reappearing for screening. RESULTS: Data were available for 52,172 two-view mammography examinations of 24,875 women. No initial screening (prevalence) examinations were included. Cancer detection rates based on mammographic findings were 0.31% (81/25,901) for film, 0.49% (48/9841) for photon-counting, and 0.38% (63/16,430) for computed radiography. The recall rate for film was 1.4%, which was significantly higher than that for PC-DR (1.0%; P<0.001) and computed radiography (1.0%; P<0.001). The positive predictive value was lower for film (22%) than for photon-counting (47%; P<0.001) and computed radiography (39%; P<0.001). In addition, the average glandular dose was 1.1mGy for film, 0.28mGy for photon-counting and 0.92mGy for computed radiography. Thus, photon-counting provided a 75% dose reduction, and computed radiography a 16% dose reduction, over film. CONCLUSIONS: Digital radiography, especially photon-counting, performs as well as or better than screen-film radiography. Given the advantages related to improved data storage and communication, digital radiography seems to be a valid alternative to screen-film radiography.
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Mamografía/métodos , Tamizaje Masivo/métodos , Intensificación de Imagen Radiográfica/métodos , Pantallas Intensificadoras de Rayos X , Adulto , Anciano , Mama/efectos de la radiación , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Femenino , Estudios de Seguimiento , Humanos , Almacenamiento y Recuperación de la Información , Persona de Mediana Edad , Vigilancia de la Población , Valor Predictivo de las Pruebas , Dosis de Radiación , Sistemas de Información Radiológica , Estudios Retrospectivos , Suecia , Tomografía Computarizada por Rayos X/métodosAsunto(s)
Biomarcadores/análisis , Hemoglobina Fetal/análisis , Preeclampsia , Femenino , Humanos , Intercambio Materno-Fetal , Modelos Biológicos , Estrés Oxidativo , Placenta/metabolismo , Placenta/patología , Circulación Placentaria , Preeclampsia/sangre , Preeclampsia/diagnóstico , Preeclampsia/etiología , Preeclampsia/terapia , Valor Predictivo de las Pruebas , Embarazo , Primer Trimestre del EmbarazoRESUMEN
To examine reciprocal or unilateral implications between two cell destruction processes, autophagy and apoptosis, in 5-Fluorouracil (5-FU)-treated tumor cells, a combination of chemical inhibitors, RNAi and genetic approaches were used. In contrast to cancer cells harboring obstructed apoptosis, either at the DISC or the mitochondrial level, p53-deficiency generated signs of autophagy deregulation upon chemotherapy. On the other, hand disruption of lysosomal function by chloroquine, caused a profound decrease in apoptotic markers appearing in response to 5-FU. DR5, which is essential for 5-FU-induced apoptosis, accumulated in lysosomes and autophagosomes upon chloroquine treatment. Since neither 3-MA, RNAi of critical autophagy regulators or inhibition of cathepsins reversed apoptosis in a similar manner, it is likely that not autophagy per se but rather correct receptor transport is an important factor for 5-FU cytotoxicity. We found that apoptosis generated by TRAIL, the cognate ligand for DR5, remained unchanged upon chloroquine lysosomal interference, indicating that 5-FU activates the receptor by a discrete mechanism. In support, depletion of membrane cholesterol or hampering cholesterol transport drastically reduced 5-FU cytotoxicity. We conclude that targeting of lysosomes by chloroquine deregulates DR5 trafficking and abrogates 5-FU- but not TRAIL-stimulated cell elimination, hence suggesting a novel mechanism for receptor activation.