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1.
Med Mycol J ; 61(3): 33-48, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32863327

RESUMO

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances (CADS) such as the hot water extract of C. albicans and Candida water-soluble fractions (CAWS) induce coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the mannoprotein fractions (MN fractions) of clinically isolated Candida species induce vasculitis in mice. We prepared MN fractions from 26 strains of Candida species by conventional hot water extraction and compared vasculitis in DBA/2 mice. The results obtained revealed that the induction of vasculitis and resulting heart failure were significantly dependent on the species; namely, death rates on day 200 were as follows: Candida krusei (100%), Candida albicans (84%), Candida dubliniensis (47%), Candida parapsilosis (44%), Candida glabrata (32%), Candida guilliermondii (20%), and Candida tropicalis (20%). Even for C. albicans, some strains did not induce vasculitis. The present results suggest that MN-induced vasculitis is strongly dependent on the species and strains of Candida, and also that the MN fractions of some non-albicans Candida induce similar toxicity to those of C. albicans.


Assuntos
Candida albicans/química , Candida albicans/patogenicidade , Candidíase , Vasos Coronários/microbiologia , Proteínas Fúngicas/efeitos adversos , Vasculite/microbiologia , Animais , Candida albicans/classificação , Fracionamento Celular , Proteínas Fúngicas/isolamento & purificação , Camundongos Endogâmicos DBA , Especificidade da Espécie
2.
PLoS One ; 15(7): e0235793, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634162

RESUMO

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.


Assuntos
Fracionamento Celular/métodos , Células Endoteliais/química , Vesículas Extracelulares/química , Biomarcadores/análise , Centrifugação/métodos , Precipitação Química , DNA/análise , Células Endoteliais/virologia , Vesículas Extracelulares/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas/análise , RNA/análise
3.
PLoS One ; 15(7): e0235563, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645092

RESUMO

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. We performed western blotting for transmembrane iron transporter proteins; SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.


Assuntos
Western Blotting/métodos , Proteínas de Transporte de Cátions/análise , Fracionamento Celular/métodos , Células A549 , Anticorpos/imunologia , Células CACO-2 , Proteínas de Transporte de Cátions/imunologia , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Limite de Detecção , Células MCF-7
4.
Nucleic Acids Res ; 48(11): 5859-5872, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421779

RESUMO

Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization.


Assuntos
Fracionamento Celular , Microambiente Celular , Espaço Intracelular/química , RNA/análise , RNA/metabolismo , Análise de Sequência de RNA , Transcriptoma , Animais , Extratos Celulares/química , Centrifugação com Gradiente de Concentração , Feminino , Fígado/citologia , Fígado/metabolismo , Espectrometria de Massas , Camundongos , RNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Sacarose
5.
Nucleic Acids Res ; 48(9): 4725-4740, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32313943

RESUMO

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fracionamento Celular , Linhagem Celular Tumoral , Humanos , Estresse Oxidativo , Biossíntese de Proteínas , Estabilidade de RNA
6.
PLoS One ; 15(4): e0226862, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287270

RESUMO

SESN2 is a member of the evolutionarily conserved sestrin protein family found in most of the Metazoa species. The SESN2 gene is transcriptionally activated by many stress factors, including metabolic derangements, reactive oxygen species (ROS), and DNA-damage. As a result, SESN2 controls ROS accumulation, metabolism, and cell viability. The best-known function of SESN2 is the inhibition of the mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays a central role in support of cell growth and suppression of autophagy. SESN2 inhibits mTORC1 activity through interaction with the GATOR2 protein complex preventing an inhibitory effect of GATOR2 on the GATOR1 protein complex. GATOR1 stimulates GTPase activity of the RagA/B small GTPase, the component of RagA/B:RagC/D complex, preventing mTORC1 translocation to the lysosomes and its activation by the small GTPase Rheb. Despite the well-established role of SESN2 in mTORC1 inhibition, other SESN2 activities are not well-characterized. We recently showed that SESN2 could control mitochondrial function and cell death via mTORC1-independent mechanisms, and these activities might be explained by direct effects of SESN2 on mitochondria. In this work, we examined mitochondrial localization of SESN2 and demonstrated that SESN2 is located on mitochondria and can be directly involved in the regulation of mitochondrial functions.


Assuntos
Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Células A549 , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Fracionamento Celular , Respiração Celular , Citosol/metabolismo , Humanos , Espécies Reativas de Oxigênio
7.
Neuron ; 106(1): 90-107.e13, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32059759

RESUMO

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift toward a more cytosolic proteome in C9-HRE cells. Among these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient induced pluripotent stem cell (iPSC) neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS and/or FTD.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de Drosophila/genética , Demência Frontotemporal/genética , Neurônios/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/genética , Fatores de Terminação de Peptídeos/genética , RNA Mensageiro/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteína C9orf72/metabolismo , Fracionamento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Membrana Nuclear , Terminação Traducional da Cadeia Peptídica/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteoma , Frações Subcelulares , Espectrometria de Massas em Tandem
8.
Blood Cells Mol Dis ; 80: 102372, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31710879

RESUMO

The marked pro-thrombotic tendency in PNH is likely to be at least partly due to the population of platelets derived from the abnormal stem cell clone. However, identification of GPI (-) platelets by flow cytometry can be technically difficult. Here we describe a technique that involves the addition of aspirin immediately after the separation of platelet rich plasma and the use of gel filtration to isolate platelets away from plasma proteins and other blood cells. In a study of 92 analyses of samples from 50 patients, we have demonstrated that the percentage of PNH platelets correlates well with the percentage of PNH granulocytes. We also provide data on several cases where there was an extreme discrepancy between the proportion of PNH granulocytes and red cells; in these cases, the demonstration of abnormal platelets suggests that the patient is likely to be at risk of thrombosis. We believe this test will be potentially useful in the evaluation of samples from such patients and may serve as a tool to investigate the causes of hypercoagulability in PNH.


Assuntos
Plaquetas/metabolismo , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/diagnóstico , Biomarcadores , Coagulação Sanguínea , Fracionamento Celular/métodos , Eritrócitos/metabolismo , Citometria de Fluxo/métodos , Granulócitos/metabolismo , Hemoglobinúria Paroxística/complicações , Humanos , Testes de Função Plaquetária , Curva ROC , Trombose/etiologia
9.
J Nanobiotechnology ; 17(1): 119, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801555

RESUMO

The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers as possible, (ii) better understand the biologically active proteins in EVs and (iii) evaluate the resulting protein signatures in EV-activated neurons. The EV functional properties were also evaluated in neurite outgrowth assays on rat primary neurons and the RNAseq analysis of the microglia-derived EVs was performed to propose the most representative miRNAs in microglia-derived EVs. This strategy allowed validating the EV isolation, identify major biological pathways in EVs and corroborate the regenerative process in EV-activated neurons. In parallel, six different miRNAs were originally identified in microglia-derived EVs including 3 which were only known in plants until now. The analysis of the neuronal proteins under the microglial EV activation suggested possible miRNA-dependent regulation mechanisms. Taken together, this combination of methodologies showed the leech microglial EVs as neuroprotective cargos across species and contributed to propose original EV-associated miRNAs whose functions will have to be evaluated in the EV-dependent dialog between microglia and neurons.


Assuntos
Vesículas Extracelulares/genética , MicroRNAs/genética , Microglia/citologia , Animais , Fracionamento Celular , Células Cultivadas , Cromatografia em Gel , Sanguessugas/citologia , Sanguessugas/genética , Microglia/metabolismo , Neuroproteção , Ratos , Ratos Wistar , Transcriptoma , Ultracentrifugação
10.
Int J Mol Sci ; 20(19)2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31546622

RESUMO

A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of "liquid biopsy." Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.


Assuntos
Biomarcadores/metabolismo , Exossomos/metabolismo , Comunicação Celular , Fracionamento Celular , Sistemas de Liberação de Medicamentos , Exossomos/genética , Humanos , Biópsia Líquida , Neoplasias/tratamento farmacológico
11.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561474

RESUMO

Small extracellular vesicles (sEVs) mediate cell-to-cell communication. We recently reported that circulating sEVs regulate systolic blood pressure in an animal model of human systemic hypertension. However, the underlying mechanisms still remain to be elucidated. As the first step for detailed analyses, we sought to increase the yield and purity of sEVs isolated from rat plasma. We compared the concentration and size distribution of sEVs as well as protein expression of the sEV marker and contaminants among plasma sEVs isolated by the ultracentrifugation (UC) method, the precipitation with polyethylene-glycol and ultracentrifugation (PEG-UC) method, or the precipitation with polyethylene-glycol (PEG) method. Effects of anticoagulants were also examined. The total concentration of plasma sEVs isolated by the PEG or PEG-UC method was much higher than that of the UC method. In the plasma sEVs isolated by the PEG-UC method, contaminating proteins were lower, while the protein expression of certain sEV markers was higher than that of the PEG method. There was no significant difference in total concentration or protein expression of sEV markers in sEVs isolated from rat plasma treated with three different anticoagulants (heparin, ethylenediaminetetraacetic acid, or acid citrate dextrose buffer) by the PEG-UC method. We, for the first time, determined that the PEG-UC method was optimal for sEV isolation from rat plasma.


Assuntos
Vesículas Extracelulares/metabolismo , Frações Subcelulares , Animais , Biomarcadores , Fracionamento Celular , Fracionamento Químico/métodos , Humanos , Masculino , Tamanho da Partícula , Plasma , Ratos
12.
Future Med Chem ; 11(10): 1225-1236, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31280675

RESUMO

Exosomes are secreted by mammalian cells and are widely distributed in cellular systems. They are a medium of information and material transmission. The complexity of exosome nature and function is not thoroughly understood. Nevertheless, they are being confirmed as mediators of intercellular communication and play significant roles in many physiological and pathological processes. Significant obstacles to the efficient and robust isolation of large quantities of pure and specific exosomes still exist. These include a lack of understanding of the relationship between exosome characteristics and function, and a shortage of scalable solutions to separate specific exosomes from other large entities remain. Hence, generic production platforms are desired. While solutions suitable for exosome manufacturing under GMP are available, most have been developed for other purposes.


Assuntos
Técnicas de Cultura de Células/métodos , Exossomos/metabolismo , Animais , Reatores Biológicos , Comunicação Celular , Técnicas de Cultura de Células/instrumentação , Fracionamento Celular/instrumentação , Fracionamento Celular/métodos , Desenho de Equipamento , Exossomos/química , Exossomos/patologia , Humanos
13.
Cells ; 8(7)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311206

RESUMO

The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality.


Assuntos
Fracionamento Celular/métodos , Exossomos/química , Animais , Exossomos/metabolismo , Exossomos/ultraestrutura , Humanos , Proteômica/métodos
14.
PLoS One ; 14(7): e0220102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31335892

RESUMO

The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.


Assuntos
Fracionamento Celular/métodos , DNA Bacteriano/normas , Técnicas de Diagnóstico Molecular/métodos , Fracionamento Celular/economia , Fracionamento Celular/normas , DNA Bacteriano/química , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas
15.
BMC Bioinformatics ; 20(1): 360, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253078

RESUMO

BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.


Assuntos
Fracionamento Celular/métodos , Microscopia/métodos , Algoritmos , Humanos , Processamento de Imagem Assistida por Computador
16.
Int J Mol Sci ; 20(12)2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248089

RESUMO

Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Paclitaxel/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Fracionamento Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteoma , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
17.
Methods Mol Biol ; 1982: 75-101, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172467

RESUMO

The NADPH oxidase NOX2 complex consists of assembled cytosolic and redox membrane proteins. In mammalian cells, natural arachidonic acid (cis-AA), released by activated phospholipase-A2, plays an important role in the activation of the NADPH oxidase, but the mechanism of action of cis-AA is still a matter of debate. In cell-free systems, cis-AA is commonly used for activation although its structural effects are still unclear. Undoubtedly cis-AA participates in the synergistic multi-partner assembly that can be hardly studied at the molecular level in vivo due to cellular complexity. The capacity of this anionic amphiphilic fatty acid to activate the oxidase is mainly explained by its ability to disrupt intramolecular bonds, mimicking phosphorylation events in cell signaling and therefore allowing protein-protein interactions. Interestingly the geometric isomerism of the fatty acid and its purity are crucial for optimal superoxide production in cell-free assays. Indeed, optimal NADPH oxidase assembly was hampered by the substitution of the cis form by the trans forms of AA isomers (Souabni et al., BBA-Biomembranes 1818:2314-2324, 2012). Structural analysis of the changes induced by these two compounds, by circular dichroism and by biochemical methods, revealed differences in the interaction between subunits. We describe how the specific geometry of AA plays an important role in the activation of the NOX2 complex.


Assuntos
Ácido Araquidônico/metabolismo , NADPH Oxidases/metabolismo , Fagócitos/enzimologia , Ácido Araquidônico/química , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Sistema Livre de Células , Colorimetria , Ativação Enzimática , Isomerismo , Estrutura Molecular , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/enzimologia , Fagócitos/imunologia , Proteínas Recombinantes de Fusão , Análise Espectral
18.
Methods Mol Biol ; 1982: 103-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172468

RESUMO

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2 ˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2 ˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.


Assuntos
Sistema Livre de Células , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , NADPH Oxidase 5/antagonistas & inibidores , Fracionamento Celular , Membrana Celular/enzimologia , Sistema Livre de Células/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/química , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , NADPH Oxidase 5/química , NADPH Oxidase 5/genética , NADPH Oxidase 5/metabolismo , Oxirredução , Espécies Reativas de Oxigênio , Bibliotecas de Moléculas Pequenas , Análise Espectral
19.
Methods Mol Biol ; 1982: 461-472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172489

RESUMO

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.


Assuntos
Fracionamento Celular , Endossomos/metabolismo , NADPH Oxidases/metabolismo , Oxirredução , Fracionamento Celular/métodos , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Ativação Enzimática , Humanos , Espécies Reativas de Oxigênio/metabolismo
20.
J Vis Exp ; (146)2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-31058903

RESUMO

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver. Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.


Assuntos
Fracionamento Celular/métodos , Gotículas Lipídicas , Fígado/ultraestrutura , Organelas , Animais , Retículo Endoplasmático , Feminino , Lisossomos , Camundongos , Camundongos Endogâmicos C57BL , Fosfolipídeos/metabolismo , Proteínas/metabolismo
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