Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 415
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 10(1): 1313, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899020

RESUMO

Individual cells in clonal populations often respond differently to environmental changes; for binary phenotypes, such as cell death, this can be measured as a fractional response. These types of responses have been attributed to cell-intrinsic stochastic processes and variable abundances of biochemical constituents, such as proteins, but the influence of organelles is still under investigation. We use the response to TNF-related apoptosis inducing ligand (TRAIL) and a new statistical framework for determining parameter influence on cell-to-cell variability through the inference of variance explained, DEPICTIVE, to demonstrate that variable mitochondria abundance correlates with cell survival and determines the fractional cell death response. By quantitative data analysis and modeling we attribute this effect to variable effective concentrations at the mitochondria surface of the pro-apoptotic proteins Bax/Bak. Further, our study suggests that inhibitors of anti-apoptotic Bcl-2 family proteins, used in cancer treatment, may increase the diversity of cellular responses, enhancing resistance to treatment.


Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Anexina A5/química , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Corantes Fluorescentes/química , Variação Genética , Células HeLa , Humanos , Células Jurkat , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Genéticos , Compostos Orgânicos/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
2.
Anticancer Res ; 38(12): 6669-6672, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30504375

RESUMO

BACKGROUND/AIM: Annexin V and propidium iodide (PI) dual staining is commonly applied in bioscience as a method to detect apoptosis. However, excessive handling of adherent cells may interfere with the integrity of plasma membrane and hence impede the accuracy of this method. Here, we exploited PI uptake as an indicator of cell integrity and investigated how cell harvesting methods and solutions involved in common apoptosis detection techniques affected measurement results. MATERIALS AND METHODS: Different cell harvesting techniques, staining with PI and flow cytometry were performed. RESULTS: Non-fixed scrapped cells revealed significantly higher fractions of PI-positive staining compared to non-fixed trypsinized cells. In the case of harvesting cells by scrapping, samples stained in binding buffer (68.30±3.55%) showed consistently higher PI-positive staining than samples stained in PBS (36.37±5.90%) in a significant manner (p=0.015). CONCLUSION: Enzymatic harvesting using 0.25% trypsin instead of mechanical harvesting by rubber scraper caused less damage of cell integrity. Furthermore, the binding buffer used in the apoptosis detection protocol aggravated the existing plasma membrane damage caused by the rubber scraper.


Assuntos
Apoptose/fisiologia , Bioensaio/métodos , Adesão Celular/fisiologia , Separação Celular/métodos , Anexina A5/química , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Propídio/química , Coloração e Rotulagem/métodos , Tripsina/farmacologia
3.
Langmuir ; 34(34): 10040-10047, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30063356

RESUMO

Apoptosis plays a critical role in many biological processes and the etiology of various diseases of the immune system. The study of apoptosis would allow both improving the diagnosis of certain diseases and serving as a target of drug screening. In this paper, we developed a sensitive assay of single-cell apoptosis using semiconductor quantum dots (QDs) as fluorescent-labeling probes. The principle of this assay is based on the detection of phosphatidylserine (PS) exposed on the plasma membrane during the drug-induced apoptosis. The QD-labeled annexin V (AV) was prepared to specifically target PS on the membrane of apoptotic cells, and PS was detected by fluorescent imaging, flow cytometer, and single-molecule fluorescence correlation spectroscopy (FCS). We developed the procedures for conjugation of QDs to AV and for purification of their conjugates by gel chromatography. The obtained conjugates were characterized by FCS, capillary electrophoresis, and zeta potential analyzer. We studied the nonspecific adsorption of cells to different surface-modified QDs and found that the nonspecific adsorption effects were significantly reduced by modification of QDs with polyethylene glycol in the detection of apoptosis. In this assay, the results obtained by flow cytometry were consistent with the commercial test kit. Furthermore, a home-built single-molecule FCS system was developed for in situ study the drug-induced apoptosis. We observed the significant change in the diffusion coefficients of QDs on cells during the progress of cell apoptosis. Compared with conventional methods, the fluorescent methods represented here possess high sensitivity because of the use of high photo stability and brightness QDs as labeling probes and provide the temp-spatial information on a single apoptotic cell.


Assuntos
Anexina A5/química , Apoptose/fisiologia , Corantes Fluorescentes/química , Polietilenoglicóis/química , Pontos Quânticos/química , Adsorção , Apoptose/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Citometria de Fluxo/métodos , Fluorescência , Humanos , Microscopia de Fluorescência/métodos , Fosfatidilserinas/metabolismo
4.
Sci Rep ; 8(1): 12914, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150684

RESUMO

Hepatocellular carcinoma (HCC) has been recognized worldwide as one of the major causes of cancer death. The medicinal fungus Antrodia cinnamomea (A. cinnamomea) has been served as a functional food for liver protection. The aim of the present study was to investigate the potential activity of A. cinnamomea extracts as a safe booster for the anticancer activity of sorafenib, a multi-kinase inhibitor approved for the treatment of HCC. The biologically active triterpenoids in the ethanolic extracts of A. cinnamomea (EAC) were initially identified by HPLC/LC/MS then the different extracts and sorafenib were assessed in vitro and in vivo. EAC could effectively sensitize HCC cells to low doses of sorafenib, which was perceived via the ability of the combination to repress cell viability and to induce cell cycle arrest and apoptosis in HCC cells. The ability of EAC to enhance sorafenib activity was mediated through targeting mitogen-activated protein (MAP) kinases, modulating cyclin proteins expression and inhibiting cancer cell invasion. Moreover, the proposed combination significantly suppressed ectopic tumor growth in mice with high safety margins compared to single-agent treatment. Thus, this study highlights the advantage of combining EAC with sorafenib as a potential adjuvant therapeutic strategy against HCC.


Assuntos
Antrodia/química , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Animais , Anexina A5/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Células Hep G2 , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Propídio/química , Sorafenibe/química , Sorafenibe/uso terapêutico , Cicatrização/efeitos dos fármacos
5.
Sci Rep ; 8(1): 11838, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087440

RESUMO

Giant unilamellar vesicles (GUVs) are increasingly used as a versatile research tool to investigate membrane structure, morphology and phase state. In these studies, GUV preparation is typically enhanced by an externally applied electric field, a process called electroformation. We find that upon osmotic deflation, GUVs electroformed from charged and neutral lipids exhibit inward pointing lipid nanotubes, suggesting negative spontaneous curvature of the membrane. By quenching a fluorescent analog of the charged lipid, zeta potential measurements and experiments with the lipid marker annexin A5, we show that electroformed GUVs exhibit an asymmetric lipid distribution across the bilayer leaflets. The asymmetry is lost either after storing electroformed GUVs at room temperature for one day or by applying higher voltages and temperatures during electroformation. GUVs having the same lipid composition but grown via gel-assisted swelling do not show asymmetric lipid distribution. We discuss possible mechanisms for the generation and relaxation of lipid asymmetry, as well as implications for studies using electroformed vesicles. The observed effects allow to control the molecular assembly of lipid bilayer leaflets. Vesicle tubulation as reported here is an example of protein-free reshaping of membranes and is caused by compositional lipid asymmetry between leaflets.


Assuntos
Técnicas Eletroquímicas/métodos , Bicamadas Lipídicas/química , Nanotubos/química , Lipossomas Unilamelares/química , Anexina A5/química , Anexina A5/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Técnicas Eletroquímicas/instrumentação , Bicamadas Lipídicas/metabolismo , Microscopia Confocal , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Lipossomas Unilamelares/metabolismo
6.
Clin Rheumatol ; 37(12): 3359-3364, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29948348

RESUMO

Vascular antiphospholipid syndrome (VAPS) and obstetric (OAPS) are different entities because some patients only develop thrombosis (without recurrent pregnancy losses) and vice versa. Only two articles have reported that low 25-hydroxy-cholecalciferol (vitamin D3, VD3) levels were not correlated with the presence of conventional antiphospholipid antibodies (aPL Abs: anticardiolipin (aCL), anti-beta2glycoprotein I (aß2gpI), and lupus anticoagulant (LA)), but no article analyzed the association of VD3 and anti-annexin A5 (aanxA5) Abs. The aim of our study was to investigate the association between VD3, multiple positivity of conventional aPL and aanxA5 Abs levels only in female OAPS vs. VAPS. Our study included 62 consecutive female PAPS patients. Concentrations of Abs were measured by ELISA, while VD3 levels were determined by immunochemiluminescence. Only 10/62 (16.13%) had sufficient (≥ 30 ng/ml) VD3 levels, while 48/62 (77.42%) and 4/62 (6.45%) had insufficiency and VD3 deficiency, respectively. Statistically significant VD3 deficiency was noticed in VAPS (vs. OAPS, P = 0.013). A negative correlation between VD3 levels and the age of patients was noticed (r = - 0.493, P = 0.032) only in VAPS subgroup. Multiple positivity of aPL and aanxA5 Abs was not associated with VD3 deficiency. Newly emerging aPL Abs, such as aanxA5 Abs, or their combinations with classical aPL Abs are not associated with VD3 deficiency in neither OAPS nor VAPS patients. Due to its immunomodulatory roles in B-Ly homeostasis, supplementation with VD3 should be considered in APS, at least in subgroup with severe form of the disease, i.e., VAPS.


Assuntos
Anexina A5/química , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Calcifediol/química , Aborto Habitual , Adulto , Anexina A5/imunologia , Anticorpos Anticardiolipina/imunologia , Síndrome Antifosfolipídica/sangue , Calcifediol/imunologia , Cardiolipinas/imunologia , Colecalciferol/sangue , Colecalciferol/deficiência , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Inibidor de Coagulação do Lúpus/imunologia , Pessoa de Meia-Idade , Gravidez , Estudos Retrospectivos , Trombose/imunologia , Trombose/patologia , beta 2-Glicoproteína I/imunologia
7.
Atherosclerosis ; 272: 118-128, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29602139

RESUMO

Atherosclerotic plaque rupture is the primary mechanism responsible for myocardial infarction and stroke, the top two killers worldwide. Despite being potentially fatal, the ubiquitous prevalence of atherosclerosis amongst the middle aged and elderly renders individual events relatively rare. This makes the accurate prediction of MI and stroke challenging. Advances in imaging techniques now allow detailed assessments of plaque morphology and disease activity. Both CT and MR can identify certain unstable plaque characteristics thought to be associated with an increased risk of rupture and events. PET imaging allows the activity of distinct pathological processes associated with atherosclerosis to be measured, differentiating patients with inactive and active disease states. Hybrid integration of PET with CT or MR now allows for an accurate assessment of not only plaque burden and morphology but plaque biology too. In this review, we discuss how these advanced imaging techniques hold promise in redefining our understanding of stable and unstable coronary artery disease beyond symptomatic status, and how they may refine patient risk-prediction and the rationing of expensive novel therapies.


Assuntos
Diagnóstico por Imagem/métodos , Diagnóstico por Imagem/tendências , Placa Aterosclerótica/diagnóstico por imagem , Anexina A5/química , Apoptose , Aterosclerose/diagnóstico por imagem , Doença da Artéria Coronariana/diagnóstico por imagem , Fluordesoxiglucose F18 , Radioisótopos de Gálio , Humanos , Hipóxia , Inflamação , Ligantes , Imagem por Ressonância Magnética , Infarto do Miocárdio/diagnóstico por imagem , Neovascularização Fisiológica , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Medição de Risco , Acidente Vascular Cerebral/diagnóstico por imagem , Tomografia Computadorizada por Raios X
8.
Thromb Haemost ; 118(1): 90-102, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29304529

RESUMO

Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V-containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor-annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide-annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10-100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 µg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2ß) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.


Assuntos
Músculos Abdominais/fisiopatologia , Anexina A5/química , Endotélio/lesões , Tromboelastografia , Terapia Trombolítica , Inibidor da Tripsina de Soja de Kunitz/química , Animais , Anexina A5/uso terapêutico , Anticoagulantes/química , Coagulação Sanguínea/efeitos dos fármacos , Endotélio/patologia , Fator VIIa/química , Fator Xa/química , Humanos , Radioisótopos do Iodo/química , Masculino , Peptídeos/química , Perfusão , Inibidores de Proteases/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Espectroscopia de Luz Próxima ao Infravermelho , Trombose/fisiopatologia , Inibidor da Tripsina de Soja de Kunitz/uso terapêutico
9.
BMC Complement Altern Med ; 18(1): 37, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29378549

RESUMO

BACKGROUND: Our team has identified 17 Boreal forest species from the traditional pharmacopeia of the Eastern James Bay Cree that presented promising in vitro and in vivo biological activities in the context of type 2 diabetes (T2D). We now screened the 17 plants extracts for potential anti-apoptotic activity in cultured kidney cells and investigated the underlying mechanisms. METHODS: MDCK (Madin-Darnby Canine Kidney) cell damage was induced by hypertonic medium (700 mOsm/L) in the presence or absence of maximal nontoxic concentrations of each of the 17 plant extracts. After 18 h' treatment, cells were stained with Annexin V (AnnV) and Propidium iodide (PI) and subjected to flow cytometry to assess the cytoprotective (AnnV-/PI-) and anti-apoptotic (AnnV+/PI-) potential of the 17 plant extracts. We then selected a representative subset of species (most cytoprotective, moderately so or neutral) to measure the activity of caspases 3, 8 and 9. RESULTS: Gaultheria hispidula and Abies balsamea are amongst the most powerful cytoprotective and anti-apoptotic plants and appear to exert their modulatory effect primarily by inhibiting caspase 9 in the mitochondrial apoptotic signaling pathway. CONCLUSION: We conclude that several Cree antidiabetic plants exert anti-apoptotic activity that may be relevant in the context of diabetic nephropathy (DN) that affects a significant proportion of Cree diabetics.


Assuntos
Hipoglicemiantes/farmacologia , Medicina Tradicional , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Substâncias Protetoras/farmacologia , Animais , Anexina A5/química , Apoptose/efeitos dos fármacos , Canadá , Caspases/metabolismo , Nefropatias Diabéticas/metabolismo , Cães , Hipoglicemiantes/química , Células Madin Darby de Rim Canino , Extratos Vegetais/química , Propídio/química , Substâncias Protetoras/química
10.
Mol Imaging Biol ; 20(2): 249-259, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28785938

RESUMO

PURPOSE: Apoptosis is a key factor in unstable plaques. The aim of this study is to evaluate the utility of visualizing atherosclerotic plaques with radiolabeled duramycin and Annexin V. PROCEDURES: ApoE-/- mice were fed with a high-fat diet to develop atherosclerosis, C57 mice as a control. Using a routine conjugation protocol, highly pure [99mTc]duramycin and [99mTc]Annexin V were obtained, which were applied for in vitro cell assays of apoptosis and in vivo imaging of atherosclerotic plaques in the animal model. Oil Red O staining, TUNEL, hematoxylin-eosin (HE), and CD68 immunostaining were used to evaluate the deposition of lipids and presence of apoptotic macrophages in the lesions where focal intensity positively correlated with the uptake of both tracers. RESULTS: [99mTc]duramycin and [99mTc]Annexin V with a high radiochemical purity (97.13 ± 1.52 and 94.94 ± 0.65 %, respectively) and a well stability at room temperature were used. Apoptotic cells binding activity to [99mTc]duramycin (Kd, 6.92 nM and Bmax, 56.04 mol/1019 cells) was significantly greater than [99mTc]Annexin V (Kd, 12.63 nM and Bmax, 31.55 mol/1019 cells). Compared with [99mTc]Annexin V, [99mTc]duramycin bound avidly to atherosclerotic lesions with a higher plaque-to-background ratio (P/B was 8.23 ± 0.91 and 5.45 ± 0.48 at 20 weeks, 15.02 ± 0.23 and 12.14 ± 0.22 at 30 weeks). No plaques were found in C57 control mice. Furthermore, Oil Red O staining showed lipid deposition areas were significantly increased in ApoE-/- mice at 20 and 30 weeks, and TUNEL and CD68 staining confirmed that the focal uptake of both tracers contained abundant apoptotic macrophages. CONCLUSIONS: This stable, fast clearing, and highly specific [99mTc]duramycin, therefore, can be useful for the quantification of vulnerable atherosclerotic plaques.


Assuntos
Anexina A5/química , Bacteriocinas/química , Compostos de Organotecnécio/química , Placa Aterosclerótica/diagnóstico por imagem , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Apoptose , Humanos , Lipídeos/química , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Compostos Radiofarmacêuticos/química
11.
J Am Chem Soc ; 139(50): 18365-18375, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29206031

RESUMO

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.


Assuntos
Alanina/análogos & derivados , Albuminas/química , Aminas/química , Anexina A5/química , Sinaptotagmina I/química , Alanina/química , Animais , Anticorpos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Crizotinibe , Dissulfetos/química , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Cinética , Camundongos , Modelos Moleculares , Estrutura Molecular , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Teoria Quântica
12.
Molecules ; 22(12)2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29257055

RESUMO

Background: Although domain IV of annexin A5 (anxA5) may be less effective in binding phosphatidylserine (PS), the four domains together may guarantee the maximum binding of anxA5 to the PS membrane. Additionally, previous research has shown that annexin mutants lacking one or more domain(s) have different biological activities compared to the wild-type. The present research mainly aims to study the role of domain IV in the crucial PS-binding function of anxA5. Methods: The domain IV-truncated anxA5 protein was constructed and purified. Isothermal titration calorimetry, flow cytometry and activated partial thromboplastin time were adopted to examine the function of domain IV in anxA5-PS binding directly or indirectly. Results: The domain IV-truncated form of anxA5 is impaired in binding PS liposome and apoptotic cells, and anticoagulation activity. The mutant cannot bind calcium, but binds PS only in the presence of calcium. Conclusions: Truncation of domain IV of anxA5 destroys its calcium-binding ability and impairs its PS-binding activity. Truncation of domain IV may induce conformation change of anxA5 or reduce the hydrophobic interactions between protein and membrane, which may explain the decrease of PS-binding affinity of the mutant.


Assuntos
Anexina A5/química , Cálcio/química , Fosfatidilserinas/química , Anexina A5/fisiologia , Apoptose , Sítios de Ligação , Membrana Celular , Humanos , Células Jurkat , Fosfatidilserinas/metabolismo , Ligação Proteica
13.
Cytometry A ; 91(11): 1115-1124, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29072808

RESUMO

Annexin-V/propidium iodide method (A-V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. However, A-V/PI does not permit fixation and/or permeabilization of cells making impossible evaluation of intracellular markers, restricting the analysis in a narrow time frame after staining and excluding the possibility to study pathogen-infected cells. We developed a method permitting fixation and permeabilization of stained cells: Fixed Apoptotic/Necrotic (FAN) cells test. FAN relies on the same principle of A-V/PI, but uses reagents that maintain their binding and fluorescence characteristics after fixation/permeabilization: a fluorochrome-labeled anti-phosphatidylserine antibody and fluorescent amine-binding dyes. FAN was tested to discriminate apoptotic and necrotic cells using different stimuli on several cell types and results were always comparable to those obtained using A-V/PI. FAN, unlike A-V/PI, permitted to correlate cell death with intracellular and surface markers expression and to perform cytometry even two weeks after sample preparation. As fixation of stained cells inactivates infective pathogens, we used FAN in an in vitro model of Mycobacterium tuberculosis (Mtb) infection of macrophages to monitor cellular infection and cell death induction. Using a red-fluorescent Mtb, fluorochrome labeled anti-TNF-α and anti-MHC class II monoclonal antibodies, FAN permitted to establish that the extent of macrophage death correlates with intracellular Mtb content and that dying cells accumulate TNF-α and down-modulate MHC class II molecules. Results suggest that FAN may represent an additional tool to study programmed cell death particularly useful when fixation procedures are required for a safe infected sample analysis or to comparatively analyze multiple samples. © 2017 International Society for Advancement of Cytometry.


Assuntos
Rastreamento de Células/métodos , Citometria de Fluxo/métodos , Necrose/patologia , Anexina A5/química , Apoptose/efeitos dos fármacos , Fixadores/química , Corantes Fluorescentes/química , Humanos , Propídio/química , Coloração e Rotulagem/métodos
14.
Chembiochem ; 18(22): 2231-2235, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-28901721

RESUMO

Deregulation in apoptosis induces numerous diseases such as cancer, cardiovascular, and neurodegenerative diseases. Detection of apoptotic cells is crucial for understanding the mechanism of these diseases and for therapy development. Although optical imaging using visible-emitting fluorescent probes, such as FITC-labeled annexin V, is widely used for the detection of apoptotic cells, there are very limited probes that can be used in the near-infrared region (NIR) over 700 nm. Compared with visible light, NIR light is highly permeable in turbid biological samples and tissues. In addition, optical imaging in the NIR region shows low autofluorescence from biological samples, leading to clearer images with high signal to background ratios. Here, we report the synthesis of bioluminescence resonance energy transfer (BRET)-coupled annexin V-functionalized quantum dots (QDs) and their application to NIR optical detection of apoptotic cells.


Assuntos
Anexina A5/química , Apoptose , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Pontos Quânticos , Espectroscopia de Luz Próxima ao Infravermelho , Linhagem Celular Tumoral , Humanos
15.
J Water Health ; 15(3): 360-366, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28598340

RESUMO

Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.


Assuntos
Água Potável/química , Minerais/farmacologia , Água do Mar/análise , Purificação da Água , Animais , Anexina A5/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Corantes/química , Fluoresceína-5-Isotiocianato/química , Camundongos , Células NIH 3T3 , Propídio/química , Água do Mar/química , Coloração e Rotulagem , Sais de Tetrazólio/química , Tiazóis/química
16.
Proc Natl Acad Sci U S A ; 114(27): 7089-7094, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630342

RESUMO

The role of autoimmunity in cardiovascular (CV) diseases has been increasingly recognized. Autoimmunity is most commonly examined by the levels of circulating autoantibodies in clinical practices. Measurement of autoantibodies remains, however, challenging because of the deficiency of reproducible, sensitive, and standardized assays. The lack of multiplexed assays also limits the potential to identify a CV-specific autoantibody profile. To overcome these challenges, we developed a nanotechnology-based plasmonic gold chip for autoantibody profiling. This approach allowed simultaneous detection of 10 CV autoantibodies targeting the structural myocardial proteins, the neurohormonal regulatory proteins, the vascular proteins, and the proteins associated with apoptosis and coagulation. Autoantibodies were measured in four groups of participants across the continuum of hypertensive heart diseases. We observed higher levels of all 10 CV autoantibodies in hypertensive subjects (n = 77) compared with healthy participants (n = 30), and the autoantibodies investigated were related to each other, forming a highly linked network. In addition, we established that autoantibodies to troponin I, annexin-A5, and beta 1-adrenegic receptor best discriminated hypertensive subjects with adverse left ventricular (LV) remodeling or dysfunction (n = 49) from hypertensive subjects with normal LV structure and function (n = 28). By further linking these three significant CV autoantibodies to the innate and growth factors, we revealed a positive but weak association between autoantibodies to troponin I and proinflammatory cytokine IL-18. Overall, we demonstrated that this platform can be used to evaluate autoantibody profiles in hypertensive subjects at risk for heart failure.


Assuntos
Autoanticorpos/química , Ouro/química , Cardiopatias/diagnóstico , Hipertensão/diagnóstico , Nanopartículas Metálicas/química , Idoso , Anexina A5/química , Autoimunidade , Estudos de Casos e Controles , Ecocardiografia , Feminino , Cardiopatias/imunologia , Insuficiência Cardíaca/imunologia , Humanos , Hipertensão/imunologia , Masculino , Pessoa de Meia-Idade , Nanotecnologia , Receptores Adrenérgicos beta 1/química , Risco , Troponina I/química
17.
Sci Rep ; 7(1): 2552, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28566720

RESUMO

Human umbilical cord mesenchymal stem cells (huMSCs) can treat primary ovarian insufficiency (POI) related to ovarian granulosa cell (OGC) apoptosis caused by cisplatin chemotherapy. Exosomes are a class of membranous vesicles with diameters of 30-200 nm that are constitutively released by eukaryotic cells. Exosomes mediate local cell-to-cell communication by transferring microRNAs and proteins. In the present study, we demonstrated the effects of exosomes derived from huMSCs (huMSC-EXOs) on a cisplatin-induced OGC model in vitro and discussed the preliminary mechanisms involved in these effects. We successfully extracted huMSC-EXOs from huMSC culture supernatant and observed the effective uptake of exosomes by cells with fluorescent staining. Using flow cytometry (with annexin-V/PI labelling), we found that huMSC-EXOs increased the number of living cells. Western blotting showed that the expression of Bcl-2 and caspase-3 were upregulated, whilst the expression of Bax, cleaved caspase-3 and cleaved PARP were downregulated to protect OGCs. These results suggest that huMSC-EXOs can be used to prevent and treat chemotherapy-induced OGC apoptosis in vitro. Therefore, this work provides insight and further evidence of stem cell function and indicates that huMSC-EXOs protect OGCs from cisplatin-induced injury in vitro.


Assuntos
Cisplatino/antagonistas & inibidores , Exossomos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Substâncias Protetoras/farmacologia , Animais , Anexina A5/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Cisplatino/farmacologia , Feminino , Citometria de Fluxo , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Substâncias Protetoras/química , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
18.
Biochim Biophys Acta Biomembr ; 1859(10): 1911-1920, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28549727

RESUMO

Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes.


Assuntos
Fosfatase Alcalina/química , Fosfatase Alcalina/metabolismo , Anexina A5/química , Anexina A5/metabolismo , Proteolipídeos/química , Proteolipídeos/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Biomimética/métodos , Calcificação Fisiológica/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Colágeno Tipo II/química , Colágeno Tipo II/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Fluidez de Membrana/fisiologia , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microscopia de Força Atômica/métodos , Ratos , Serina/química , Serina/metabolismo
19.
J Thromb Haemost ; 15(7): 1412-1421, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28393472

RESUMO

Essentials Annexin A5 resistance is a mechanism for antiphospholipid (aPL) syndrome. 750 patients with history of thrombosis, pregnancy complications and controls were tested. Reduced annexin A5 anticoagulant ratios (A5R) correlate with aPL antibody multipositivity. Reduced A5R may identify patients with a propensity for thrombosis or pregnancy complications. Click to hear an ISTH Academy presentation on antiphospholipid antibody syndrome by Drs de Laat and Bertolaccini SUMMARY: Background Annexin A5 (A5) is a potent anticoagulant protein that shields anionic phospholipids from coagulation reactions. Previous studies showed that antibodies from patients with antiphospholipid (aPL) syndrome (APS) interfere with annexin A5 crystallization and anticoagulant activity. Objective The purpose of this study was to investigate whether reduced values in the annexin A5 anticoagulant ratio (A5R) assay (i.e. 'annexin A5 resistance') are associated with adverse clinical events in aPL antibody-positive patients. Patients/Methods In an initial discovery phase, a group of 679 patient samples from a 'real-world' tertiary care hospital population were tested for A5R. This was followed by a validation-phase cohort of 71 asymptomatic patients with aPL antibodies and no prior history of an adverse clinical event whose baseline samples were tested for A5R then subsequently observed for up to 4 years. Results In the discovery-phase group, we found a reduction of A5R in aPL antibody-positive patients with thrombosis and/or pregnancy complications compared with aPL antibody-negative patients and controls. In addition, reduced A5R values in both the discovery-phase group and validation-phase cohort correlated with the extent of multi-positivity for standard APS tests, which has also been shown to be associated with a risk of adverse clinical outcomes. Conclusion Reduced A5R values were associated with a multi-positivity profile in aPL antibody-positive patients within both groups and with the development of adverse clinical events.


Assuntos
Anexina A5/química , Anticorpos Antifosfolipídeos/sangue , Anticoagulantes/química , Complicações Cardiovasculares na Gravidez/sangue , Trombose/sangue , Aborto Espontâneo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anexina A5/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/imunologia , Coagulação Sanguínea , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/química , Gravidez , Complicações Cardiovasculares na Gravidez/imunologia , Trombose/imunologia , Resultado do Tratamento , Adulto Jovem
20.
Oncotarget ; 8(21): 34576-34585, 2017 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-28423667

RESUMO

Microvesicles (MVs) are carriers of molecular and oncogenic signatures present in subsets of tumor cells and tumor-associated stroma, and a focus of cancer research. Although methods to detect MVs are mature, we were concerned that the buffer used could lead to false results when quantitating MVs by flow cytometry. In this work,we detected MVs by flow cytometry withthree different solutions: water, saline, and phosphate-buffered saline (PBS). The results demonstrated that PBS, when reacted with annexin V binding buffer, produced nano-sized vesicles even when there were no MVs in the sample. No similar events occurred in the saline and water groups (P < 0.01). Annexin V positive rate increased significantly when PBS was used as the buffer, compared to saline and water. These false negative results were also observed when we quantified some markers of MVs such as CD3 and CD19. A probable explanation for these findings is the production of insoluble Ca(H2PO4)2 or Ca3PO4 from calcium in the binding buffer and phosphate in PBS. Thus, considering the osmotic pressure of water, we suggest that saline is a more suitable buffer when counting MVs by flow cytometry.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Cloreto de Sódio/química , Anexina A5/química , Biomarcadores/metabolismo , Citometria de Fluxo/métodos , Células Endoteliais da Veia Umbilical Humana , Humanos , Células K562
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA