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1.
Nat Commun ; 12(1): 2284, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863904

RESUMO

Drug resistance is a major obstacle to the treatment of most human tumors. In this study, we find that dual-specificity phosphatase 16 (DUSP16) regulates resistance to chemotherapy in nasopharyngeal carcinoma, colorectal cancer, gastric and breast cancer. Cancer cells expressing higher DUSP16 are intrinsically more resistant to chemotherapy-induced cell death than cells with lower DUSP16 expression. Overexpression of DUSP16 in cancer cells leads to increased resistance to cell death upon chemotherapy treatment. In contrast, knockdown of DUSP16 in cancer cells increases their sensitivity to treatment. Mechanistically, DUSP16 inhibits JNK and p38 activation, thereby reducing BAX accumulation in mitochondria to reduce apoptosis. Analysis of patient survival in head & neck cancer and breast cancer patient cohorts supports DUSP16 as a marker for sensitivity to chemotherapy and therapeutic outcome. This study therefore identifies DUSP16 as a prognostic marker for the efficacy of chemotherapy, and as a therapeutic target for overcoming chemoresistance in cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Mitocôndrias/efeitos dos fármacos , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Neoplasias/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Fracionamento Celular , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Fosfatases de Especificidade Dupla/análise , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/análise , Neoplasias/mortalidade , Neoplasias/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
2.
J Vis Exp ; (168)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33720114

RESUMO

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Cromatografia de Afinidade , Bactérias Gram-Positivas/genética , Análise de Sequência de RNA , Sequência de Bases , Tampões (Química) , Fracionamento Celular , Análise de Dados , Regulação Bacteriana da Expressão Gênica , Humanos , Plasmídeos/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Reprodutibilidade dos Testes , Staphylococcus aureus/genética
3.
Nat Protoc ; 16(4): 2023-2050, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33674788

RESUMO

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.


Assuntos
Túbulos Renais/crescimento & desenvolvimento , Dispositivos Lab-On-A-Chip , Organoides/crescimento & desenvolvimento , Perfusão , Técnicas de Cultura de Tecidos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fracionamento Celular , Criança , Pré-Escolar , Impedância Elétrica , Feminino , Corantes Fluorescentes/química , Humanos , Lactente , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Microfluídica , Pessoa de Meia-Idade , Ratos , Adulto Jovem
4.
Carbohydr Polym ; 261: 117887, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33766374

RESUMO

Liver glycogen is a branched glucose polymer that functions as a blood-sugar buffer in animals. Previous studies have shown that glycogen's molecular structure affects its properties. This makes it important to develop a technique that extracts and purifies a representative sample of glycogen. Here we aim to optimize the sucrose density gradient centrifugation method for preserving glycogen's molecular structure by varying the density of the sucrose solution. The preservation of glycogen's structure involves: 1) minimizing molecular damage and 2) obtaining a structurally representative sample of glycogen. The addition of a 10-minute boiling step was also tested as a means for denaturing any glycogen degrading enzymes. Lower sucrose concentrations and the introduction of the boiling step were shown to be beneficial in obtaining a more structurally representative sample, with the preservation of smaller glycogen particles and decreased glycogen chain degradation.


Assuntos
Glicogênio Hepático/química , Glicogênio Hepático/isolamento & purificação , Animais , Calibragem , Fracionamento Celular/métodos , Fracionamento Celular/normas , Fracionamento Químico/métodos , Glicogênio/química , Glicogênio/isolamento & purificação , Glicogênio/metabolismo , Fígado/química , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Masculino , Camundongos , Estrutura Molecular , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/normas
5.
J Vis Exp ; (168)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33645556

RESUMO

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often poor in cells or difficult to process. Therefore, nucleic acids need to be carefully isolated. In recent years, various methods for DNA isolation have been established for tissues from many diseases, mostly cancer. Unfortunately, genomic DNA extracted from FFPE tissues is highly degraded due to the cross-linking between nucleic acid strands and proteins, as well as random breakings in sequence. Therefore, DNA quality from these samples is markedly reduced, making it a challenge for further molecular downstream analyses. Other problems with difficult tissues are, for example, the lack of cells in calcified human atherosclerotic lesions and fatty tissue, small skin biopsies, and consequently low availability of the desired nucleic acids as it is also the case in old or fixed tissues. In our laboratories, we have established a method for DNA extraction from formalin-fixed atherosclerotic lesions, using a semi-automated isolation system. We compared this method to other commercially available extraction protocols and focused on further downstream analyses. Purity and concentration of the DNA were measured by spectrometry and fluorometry. The degree of fragmentation and overall quality were assessed. The highest DNA quantity and quality was obtained with the modified blood DNA protocol for the automated extraction system, instead of the commercial FFPE protocol. With this step-by-step protocol, DNA yields from FFPE samples were in average four times higher and fewer specimens failed the extraction process, which is critical when dealing with small-vessel biopsies. Amplicon sizes from 200-800 bp could be detected by PCR. This study shows that although DNA obtained from our FFPE tissue is highly fragmented, it can still be used for successful amplification and sequencing of shorter products. In conclusion, in our hands, the automated technology appears to be the best system for DNA extraction, especially for small FFPE tissue specimen.


Assuntos
Aterosclerose/genética , DNA/isolamento & purificação , Formaldeído/química , Inclusão em Parafina , Fixação de Tecidos , Automação , Fracionamento Celular , DNA/genética , Fragmentação do DNA , Humanos , Reação em Cadeia da Polimerase
6.
J Vis Exp ; (168)2021 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-33616101

RESUMO

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-γ, IL-12 & IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host's defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers. This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases. In this paper, we polarized the naïve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.


Assuntos
Técnicas de Cultura de Células/métodos , Polaridade Celular , Separação Celular/métodos , Glicólise , Macrófagos/citologia , Animais , Bioensaio , Fracionamento Celular , Células Cultivadas , Metabolismo Energético , Eritrócitos/citologia , Fêmur/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo
7.
Methods Mol Biol ; 2261: 411-419, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421004

RESUMO

In-depth analysis of the mitochondrial proteome can be greatly improved by analyzing isolated mitochondria instead of whole cells. However, isolation of sufficient amounts of mitochondria from cell culture has proven to be notoriously difficult due to small sample size. Thus, we have developed a reproducible, controllable, and highly customizable method to isolate high microgram to low milligram amounts of intact mitochondria from cell culture samples along with an optional density gradient purification. This chapter provides a methodological update of our approach and underlines the excellent quality and coverage of the mitochondrial proteome of crude and purified mitochondria from cultured liver cancer cell lines.


Assuntos
Fracionamento Celular , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/isolamento & purificação , Proteoma , Proteômica , Animais , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Humanos
8.
Methods Mol Biol ; 2261: 535-547, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421013

RESUMO

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.


Assuntos
Fracionamento Celular , Cloroplastos/metabolismo , Cromatografia de Afinidade , Proteínas de Plantas/análise , Proteoma , Proteômica , Arabidopsis/metabolismo , Proteínas de Arabidopsis/análise , Espectrometria de Massas , Ervilhas/metabolismo , Folhas de Planta/metabolismo
9.
Methods Mol Biol ; 2261: 563-585, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33421015

RESUMO

This chapter covers the various methods of mechanical cell disruption and tissue homogenization that are currently commercially available for processing small samples s < 1 mL) to larger multikilogram production quantities. These mechanical methods of lysing do not introduce chemicals or enzymes to the system. However, the energies required when using these "harsh," high mechanical energy methods can be enough to damage the very components being sought.The destruction of cell membranes and walls is effected by subjecting the cells (a) to shearing by liquid flow, (b) to exploding by pressure differences between inside and outside of cell, (c) to collision forces by impact of beads or paddles, or (d) a combination of these forces.Practical suggestions to optimize each method, where to acquire such equipment, and links to reference sources are included. Several novel technologies are presented.


Assuntos
Fracionamento Celular/instrumentação , Extratos de Tecidos , Animais , Extratos Celulares , Centrifugação/instrumentação , Desenho de Equipamento , Humanos , Pressão , Sonicação/instrumentação , Estresse Mecânico
10.
Methods Mol Biol ; 2222: 325-361, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301101

RESUMO

Over the years, the amount of DNA in a nucleus (genome size) has been estimated using a variety of methods, but increasingly, flow cytometry (FCM) has become the method of choice. The popularity of this technique lies in the ease of sample preparation and in the large number of particles (i.e., nuclei) that can be analyzed in a very short period of time. This chapter presents a step-by-step guide to estimating the nuclear DNA content of plant nuclei using FCM. Attempting to serve as a tool for daily laboratory practice, we list, in detail, the equipment required, specific reagents and buffers needed, as well as the most frequently used protocols to carry out nuclei isolation. In addition, solutions to the most common problems that users may encounter when working with plant material and troubleshooting advice are provided. Finally, information about the correct terminology to use and the importance of obtaining chromosome counts to avoid cytological misinterpretations of the FCM data are discussed.


Assuntos
Citometria de Fluxo/métodos , Tamanho do Genoma , Genoma de Planta , Plantas/genética , Poliploidia , Reprodução , Fracionamento Celular , Núcleo Celular/genética , Cromossomos de Plantas , Corantes Fluorescentes , Modelos Biológicos
11.
Methods Mol Biol ; 2158: 295-305, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857382

RESUMO

Exosomes are membrane-bound nano-vehicles shed by most eukaryotic cells. Exosomes contain specific proteins and RNAs from parent cells, and they play key signaling roles in cellular development, modulation, and tissue regeneration. Attempts to isolate and modify exosomes to increase their targeting efficiency to specific tissue are still in their infancy. Here, we describe generation of exosomes from biopsy, isolation of exosomes by centrifugal ultrafiltration method, and approaches for manipulation of cardiac homing exosomes by chemical engineering for the treatment of myocardial infarction.


Assuntos
Exossomos , Tecnologia Farmacêutica/métodos , Animais , Plaquetas/química , Técnicas de Cultura de Células/métodos , Fracionamento Celular/métodos , Membrana Celular/química , Membrana Celular/metabolismo , Exossomos/ultraestrutura , Humanos , Camundongos , Terapia de Alvo Molecular , Miocárdio/citologia , Peptídeos/análise , Peptídeos/metabolismo , Ratos , Ultracentrifugação , Ondas Ultrassônicas
12.
Methods Mol Biol ; 2158: 307-321, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857383

RESUMO

The adult mammalian heart's potential for regeneration is very inefficient. Importantly, adult mammalian cardiomyocytes (CMs) are characterized as a cell population with very limited mitotic potential. Conversely, the neonatal mouse heart possesses a brief, yet robust, regenerative capacity within the first week of life. Cell type-specific enrichment procedures are essential for characterizing the full spectrum of epigenomic landscapes and gene regulatory networks deployed by mammalian CMs. In this chapter, we describe a protocol useful for purifying CM nuclei from mammalian cardiac tissue. Furthermore, we detail a low-input procedure suitable for the parallel genome-wide profiling of chromatin accessibility, histone modifications, and transcription factor-binding sites. The CM nuclei purified using this process are suitable for multi-omic profiling approaches.


Assuntos
Fracionamento Celular/métodos , Núcleo Celular/química , Núcleo Celular/genética , Epigenômica/métodos , Miócitos Cardíacos/química , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Separação Imunomagnética/métodos , Camundongos , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismo
13.
Methods Mol Biol ; 2211: 147-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33336276

RESUMO

Cell-derived Drug Delivery Systems (DDSs), particularly exosomes, have grown in popularity and have been increasingly explored as novel DDSs, due to their intrinsic targeting capabilities. However, clinical translation of exosomes is impeded by the tedious isolation procedures and poor yield. Cell-derived nanovesicles (CDNs) have recently been produced and proposed as exosome-mimetics. Various methods for producing exosome-mimetics have been developed. In this chapter, we present a simple, efficient, and cost-effective CDNs production method that uses common laboratory equipment (microcentrifuge) and spin cups. Through a series of extrusion and size exclusion steps, CDNs are produced from in vitro cell culture and are found to highly resemble the endogenous exosomes. Thus, we envision that this strategy holds great potential as a viable alternative to exosomes in the development of ideal DDS.


Assuntos
Biomimética , Micropartículas Derivadas de Células , Sistemas de Liberação de Medicamentos , Exossomos , Nanopartículas , Vesículas Transportadoras , Animais , Biomarcadores , Biomimética/métodos , Fracionamento Celular/métodos , Linhagem Celular , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Fenômenos Químicos , Cromatografia em Gel , Sistemas de Liberação de Medicamentos/métodos , Exossomos/metabolismo , Exossomos/ultraestrutura , Humanos , Camundongos , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura
14.
Methods Mol Biol ; 2220: 137-153, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975772

RESUMO

Proteomics has become an essential tool to answer biologists' questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic organisms. However, not all the different cell "compartments" are easily accessible, and the analysis of cell envelope proteins is particularly challenging. For the Gram-positive bacterium Listeria monocytogenes, one of the main foodborne pathogen microorganisms, the study of surface proteins is crucial to better understand the mechanisms of pathogenicity, as well as adaptation/resistance to and persistence in hostile environments. The evolution of proteomic techniques, and particularly the possibility of separating and analyzing complex protein samples by off-gel (LC-MS/MS) versus in-gel (two-dimensional electrophoresis) approach, has opened the doors to new extraction and preparation methods to target the different subproteomes. Here, we describe three procedures to prepare and analyze intracellular, exocellular, and cell surface proteins: (1) the cell fractionation, based on cell broken and separation of protein subfractions by differential centrifugation; (2) the biotinylation, based on the labeling of cell surface proteins and their selective extraction; and (3) the enzymatic shaving by the action of trypsin on intact cells. These complementary methods allow to encompass all L. monocytogenes subproteomes for general profiling or target studies and could be applicable to other Gram-positive bacteria.


Assuntos
Proteínas de Bactérias/análise , Listeria monocytogenes/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas de Bactérias/isolamento & purificação , Biotinilação , Fracionamento Celular/métodos , Centrifugação/métodos , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Listeria monocytogenes/citologia , Listeriose/microbiologia
15.
Nucleic Acids Res ; 49(1): 400-415, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330923

RESUMO

In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Ribossomos/metabolismo , Espectrometria de Massas em Tandem/métodos , Acetilação , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Fracionamento Celular/métodos , Chlamydomonas reinhardtii/genética , Regulação da Expressão Gênica de Plantas , Separação Imunomagnética , Espectrometria de Massas , Modelos Moleculares , Acetiltransferases N-Terminal/isolamento & purificação , Acetiltransferases N-Terminal/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Subunidades Ribossômicas Maiores/metabolismo , Subunidades Ribossômicas Menores/metabolismo
16.
Methods Mol Biol ; 2187: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770499

RESUMO

Lipid rafts are microdomains on plasma membrane that contain high levels of cholesterol and sphingolipids. Because of the detergent-resistant property of lipid rafts, lipid rafts isolated by methods that use detergents frequently yield different results. Artifacts can also be introduced through the use of detergents. These limitations could be overcome with a detergent-free method which eliminates possible artificial influences. Importantly, lipid rafts prepared with a detergent-free method is more compatible to mass spectrometric analysis since the ion suppression effect is largely reduced.This chapter describes a detergent-free two-step method for preparation of lipid rafts. Firstly, a purified plasma membrane fraction is prepared from cells by sedimentation of the postnuclear supernatant (PNS) in a Percoll gradient. Secondly, the as-prepared plasma membranes are sonicated to release lipid rafts which are further isolated by flotation in a continuous gradient of Optiprep solution. Then, we introduce a typical shotgun lipidomics workflow that can be used as a cost-effective and relatively high throughput method to determine the lipidomes of lipid rafts.The method also makes an easy start for lipidomics studies.


Assuntos
Detergentes/química , Lipidômica/métodos , Microdomínios da Membrana/química , Fracionamento Celular/métodos , Colesterol/química , Espectrometria de Massas/métodos , Esfingolipídeos/química
17.
Methods Mol Biol ; 2187: 131-145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770505

RESUMO

The traditional methods to study lipid rafts and their association with membrane proteins are based mainly on the isolation of a detergent-resistant membrane by biochemical fractionation. However, the use of detergents may induce lipid segregation and/or redistribution of membrane proteins during the process of sample preparation. Here, we describe a detergent-free method to study the glycolipid and growth factor receptor interaction and their association with lipid rafts. This method combines the biochemical and immunoblotting tools with confocal microscopic imaging, which allows for evaluation and verification of the membrane protein interaction and association with the lipid rafts components in a multifaceted manner.


Assuntos
Glicolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Fracionamento Celular/métodos , Células Cultivadas , Detergentes/metabolismo , Proteínas de Membrana/metabolismo , Camundongos
18.
Methods Mol Biol ; 2187: 313-325, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32770515

RESUMO

The analysis of protein enrichment in the detergent-resistant membranes (DRMs) isolated from immune cells enables us to analyze a link between the membrane lipid dynamics and cell activation. Here, we describe the fractionation of detergent-resistant membranes and the correlative analysis of the enrichment of T cell receptor (TCR) and ω-azido-modified synthetic ceramide in those fractions upon TCR stimulation.


Assuntos
Membrana Celular/metabolismo , Esfingolipídeos/metabolismo , Fracionamento Celular/métodos , Linhagem Celular Tumoral , Células Cultivadas , Ceramidas/metabolismo , Detergentes/metabolismo , Humanos , Células Jurkat , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
19.
Nat Commun ; 11(1): 5187, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056988

RESUMO

Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.


Assuntos
Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Neurospora crassa/fisiologia , Biossíntese de Proteínas , Fracionamento Celular , Microscopia Crioeletrônica , Proteínas Fúngicas/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/ultraestrutura , Modelos Moleculares , NAD/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo
20.
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
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