Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 56.831
Filtrar
1.
Bratisl Lek Listy ; 120(9): 630-635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475544

RESUMO

OBJECTIVES: To test the hypothesis if mitochondrial bioenergetic function analyzed in circulating platelets may represent peripheral signature of mitochondrial dysfunction in nephropathy associated to non-communicable human diseases such as cardiovascular diseases, diabetes and with statins treatment. METHODS: High-resolution respirometry was used for analysis of mitochondrial bioenergetics in human platelets isolated from peripheral blood. This method is less-invasively compared to skeletal muscle biopsy. Patients with nephropathies and in combination with non-communicable diseases were included in the study. RESULTS: This pilot study showed platelet mitochondrial bioenergy dysfunction in patients with nephropathies and non-communicable diseases. Positive effect of treatment with 10 mg atorvastatin on platelet mitochondrial respiratory chain Complex I-linked respiration and ATP production in patients with nephropathies, diabetes and 80 mg atorvastatin in patient with nephropathy and dialysis was found. Positive effect of 80 mg fluvastatin treatment, and negative effect of thrombocytopenia and renal transplantation on platelet mitochondrial bioenergy was determined. CONCLUSION: High-resolution respirometry allowed detection of small changes in platelet mitochondrial function. This method could be used as a sensitive bioenergetic test of mitochondrial function for diagnosis and monitoring the therapy in patients with nephropathy (Tab. 1, Fig. 3, Ref. 39).


Assuntos
Plaquetas/metabolismo , Metabolismo Energético , Nefropatias/metabolismo , Mitocôndrias/metabolismo , Doenças não Transmissíveis , Respiração Celular , Humanos , Projetos Piloto
2.
Anticancer Res ; 39(9): 4865-4876, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519589

RESUMO

BACKGROUND/AIM: Hypoxia promotes tumor proliferation and metastasis in colorectal cancer (CRC). Since the tumor microenvironment is generally characterized by hypoxia, its understanding is important for cancer therapy. We hypothesized that hypoxia promotes the mitochondrial function, mobility, and proliferation of CRC by up-regulating peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). MATERIALS AND METHODS: To assess the effects of PGC-1α under hypoxia, we investigated the mitochondrial function, cell motility, and sphere formation as well as proliferation and apoptosis of CRC. RESULTS: Under hypoxia, we confirmed the increased expression of PGC-1α and reduced production of reactive oxygen species (ROS) by activating anti-oxidant enzymes. Also, up-regulation of PGC-1α enhanced the motility, sphere formation, and proliferation of CRC. Under the presence of the anti-cancer drug 5-fluorouracil (5FU), up-regulation of PGC-1α under hypoxia promoted resistance of CRC against 5FU-induced apoptosis. CONCLUSION: Targeting PGC-1α could to be a powerful strategy for CRC therapy.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Hipóxia/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Apoptose , Catalase/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
3.
Hum Genet ; 138(10): 1183-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31471722

RESUMO

The glutamate pyruvate transaminase 2 (GPT2) gene produces a nuclear-encoded mitochondrial enzyme that catalyzes the reversible transfer of an amino group from glutamate to pyruvate, generating alanine and alpha-ketoglutarate. Recessive mutations in GPT2 have been recently identified in a new syndrome involving intellectual and developmental disability (IDD), postnatal microcephaly, and spastic paraplegia. We have identified additional families with recessive GPT2 mutations and expanded the phenotype to include small stature. GPT2 loss-of-function mutations were identified in four families, nine patients total, including: a homozygous mutation in one child [c.775T>C (p.C259R)]; compound heterozygous mutations in two siblings [c.812A>C (p.N271T)/c.1432_1433delGT (p.V478Rfs*73)]; a novel homozygous, putative splicing mutation [c.1035C>T (p.G345=)]; and finally, a recurrent mutation, previously identified in a distinct family [c.1210C>T (p.R404*)]. All patients were diagnosed with IDD. A majority of patients had remarkably small stature throughout development, many < 1st percentile for height and weight. Given the potential biological function of GPT2 in cellular growth, this phenotype is strongly suggestive of a newly identified clinical susceptibility. Further, homozygous GPT2 mutations manifested in at least 2 of 176 families with IDD (approximately 1.1%) in a Pakistani cohort, thereby representing a relatively common cause of recessive IDD in this population, with recurrence of the p.R404* mutation in this population. Based on variants in the ExAC database, we estimated that approximately 1 in 248 individuals are carriers of moderately or severely deleterious variants in GPT2.


Assuntos
Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Genes Recessivos , Predisposição Genética para Doença , Mutação , Fenótipo , Transaminases/genética , Adolescente , Alelos , Substituição de Aminoácidos , Deficiências do Desenvolvimento/metabolismo , Ativação Enzimática , Éxons , Feminino , Frequência do Gene , Estudos de Associação Genética , Genética Populacional , Genótipo , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Imagem por Ressonância Magnética , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Linhagem , Conformação Proteica , Sítios de Splice de RNA , Análise de Sequência de DNA , Relação Estrutura-Atividade , Transaminases/química , Transaminases/metabolismo
4.
J Agric Food Chem ; 67(37): 10448-10457, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453693

RESUMO

Carabrone is isolated from Carpesium macrocephalum Franch. et Sav, which has good fungicidal activity, especially for Gaeumannomyces graminis (Get). According to previous studies, we speculated that carabrone targets the mitochondrial enzyme complex III of Get. To elucidate the mode of action, we used carabrone to induce oxidative stress and apoptosis in Get. Incubation with carabrone reduced the burst of reactive oxygen species (ROS) and mitochondrial membrane potential, as well as phosphatidylserine release. Carabrone caused ROS accumulation in mycelia by inhibiting the activity of antioxidase enzymes, among which inhibition of glutathione reductase (GR) activity was most obvious. The catalytic center of GR consists of l-cysteine residues that react with the α-methylene-γ-butyrolactone active site of carabrone. Additionally, a positive TUNEL reaction led to diffusion of the DNA electrophoresis band and upregulation of Ggmet1 and Ggmet2. We propose that carabrone inhibits antioxidant enzymes and promotes ROS overproduction, which causes membrane hyperpermeability, release of apoptotic factors, activation of the mitochondria-mediated apoptosis pathway, and fungal cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Ascomicetos/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ascomicetos/citologia , Ascomicetos/metabolismo , Asteraceae/química , Proteínas Fúngicas/metabolismo , Glutationa Redutase/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
Chem Commun (Camb) ; 55(70): 10472-10475, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31411208

RESUMO

A mitochondria-targeted photodynamic therapy (PDT) agent was designed and synthesized. Upon light irradiation, it can produce photoacid and its photolysis products can further sensitize 1O2 generation, causing dual-mode (oxygen-independent and oxygen-dependent) photodynamic damage in mitochondria and killing cancer cells effectively even under hypoxic conditions.


Assuntos
Irídio/farmacologia , Mitocôndrias/metabolismo , Fotoquimioterapia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Oxigênio Singlete/metabolismo
6.
Adv Exp Med Biol ; 1158: 1-16, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452132

RESUMO

Given the role of mitochondria in modulating many cellular functions, it is not surprising that they can play a crucial role also in molecular pathophysiology of cancer. In particular, the discovery in recent decades of a link between cancer metabolic processes, alterations of mitochondrial DNA, oncogenes and tumor suppressors has led not only to a renaissance of interest in Warburg's pioneering work, but also to a reexamination of his original observations above all in relation to the current knowledge in cancer cell metabolism. It follows that, although mitochondrial contribution to the pathogenesis of cancer has historically tended to be neglected, it is now evident that reprogrammed mitochondria can contribute to a complex bioenergetic adjustment that sustains not only tumor formation but also its progression. Most importantly, cancer cell metabolism seems to have a role in diversified aspects related to cancer pathophysiology (i.e., aggressiveness, recurrence, metastatic dissemination). Hence, it is imperative to always consider cancer cell metabolism, its adaptability, its influences but, above all, its functional heterogeneity in a single tumor, for a really rational and valid approach towards molecular biology of cancer.


Assuntos
Mitocôndrias , Neoplasias , Proteômica , Metabolismo Energético , Humanos , Mitocôndrias/metabolismo , Neoplasias/fisiopatologia , Oncogenes
7.
Adv Exp Med Biol ; 1158: 17-44, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452133

RESUMO

Parkinson's disease (PD) is a multifactorial disorder whose etiology is not completely understood. Strong evidences suggest that mitochondrial impairment and altered mitochondrial disposal play a key role in the development of this pathology. Here we show this association in both genetic and sporadic forms of the disease. Moreover, we describe the mitochondrial dysfunctions in toxin-induced models of PD, thus highlighting the importance of environmental factors in the onset of this pathology. In particular, we focus our attention on mitochondrial dynamics, mitochondrial biogenesis, and mitophagy and explain how their impairment could have a negative impact on dopaminergic neurons function and survival. Lastly, we aim at clarifying the important role played by proteomics in this field of research, proteomics being a global and unbiased approach suitable to unravel alterations of the molecular pathways in multifactorial diseases.


Assuntos
Proteínas Mitocondriais , Doença de Parkinson , Neurônios Dopaminérgicos/patologia , Humanos , Mitocôndrias/metabolismo , Degradação Mitocondrial , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/fisiopatologia
8.
Adv Exp Med Biol ; 1158: 45-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452134

RESUMO

The centrality of the mitochondrion in the evolution and control of the cellare now supported by many experimental studies. Not only with regard to the energy metabolism but also and especially with regard to the other functions indispensable for the cell such as apoptosis and the control of innate immunity through different complex cell signaling pathways. All this makes them one of the main targets during infections supported by pathogenic microorganisms. The interaction and control of these organelles by pathogens results, from the latest experimental evidence, of fundamental importance in the fate of the host cell and in the progression of infectious diseases.


Assuntos
Interações Hospedeiro-Patógeno , Mitocôndrias , Apoptose , Imunidade Inata , Mitocôndrias/metabolismo , Transdução de Sinais
9.
Adv Exp Med Biol ; 1158: 59-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452135

RESUMO

Mitochondria have a central role in cellular metabolism and reversible post-translational modifications regulate activity of mitochondrial proteins. Thanks to advances in proteomics, lysine acetylation has arisen as an important post-translational modification in the mitochondrion. During acetylation an acetyl group is covalently attached to the epsilon amino group in the side chain of lysine residues using acetyl-CoA as the substrate donor. Therefore the positive charge is neutralized, and this can affect the function of proteins thereby regulating enzyme activity, protein interactions, and protein stability. The major deacetylase in mitochondria is SIRT3 whose activity regulates many mitochondrial enzymes. The method of choice for the analysis of acetylated proteins foresees the combination of mass spectrometry-based proteomics with affinity enrichment techniques. Beyond the identification of lysine-acetylated proteins, many studies are moving towards the characterization of acetylated patterns in different diseases. Indeed, modifications in lysine acetylation status can directly alter mitochondrial function and, therefore, be linked to human diseases such as metabolic diseases, cancer, myocardial injury and neurodegenerative diseases. Despite the progress in the characterization of different lysine acetylation sites, additional studies are needed to differentiate the specific changes with a significant biological relevance.


Assuntos
Lisina , Mitocôndrias , Fenótipo , Acetilação , Humanos , Lisina/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional
10.
Adv Exp Med Biol ; 1158: 83-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452137

RESUMO

Mitochondria (mt) are double-membraned, dynamic organelles that play an essential role in a large number of cellular processes, and impairments in mt function have emerged as a causative factor for a growing number of human disorders. Given that most biological functions are driven by physical associations between proteins, the first step towards understanding mt dysfunction is to map its protein-protein interaction (PPI) network in a comprehensive and systematic fashion. While mass-spectrometry (MS) based approaches possess the high sensitivity ideal for such an endeavor, it also requires stringent biochemical purification of bait proteins to avoid detecting spurious, non-specific PPIs. Here, we outline a tagging-based affinity purification coupled with mass spectrometry (AP-MS) workflow for discovering new mt protein associations and providing novel insights into their role in mt biology and human physiology/pathology. Because AP-MS relies on the creation of proteins fused with affinity tags, we employ a versatile-affinity (VA) tag, consisting of 3× FLAG, 6 × His, and Strep III epitopes. For efficient delivery of affinity-tagged open reading frames (ORF) into mammalian cells, the VA-tag is cloned onto a specific ORF using Gateway recombinant cloning, and the resulting expression vector is stably introduced in target cells using lentiviral transduction. In this chapter, we show a functional workflow for mapping the mt interactome that includes tagging, stable transduction, selection and expansion of mammalian cell lines, mt extraction, identification of interacting protein partners by AP-MS, and lastly, computational assessment of protein complexes/PPI networks.


Assuntos
Cromatografia de Afinidade , Espectrometria de Massas , Proteínas Mitocondriais , Mapeamento de Interação de Proteínas/métodos , Fluxo de Trabalho , Animais , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/isolamento & purificação
11.
Adv Exp Med Biol ; 1158: 101-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452138

RESUMO

Targeted mass spectrometry in the selected or parallel reaction monitoring (SRM or PRM) mode is a widely used methodology to quantify proteins based on so-called signature or proteotypic peptides. SRM has the advantage of being able to quantify a range of proteins in a single analysis, for example, to measure the level of enzymes comprising a biochemical pathway. In this chapter, we will detail how to set up an SRM assay on the example of the mitochondrial protein succinate dehydrogenase [ubiquinone] flavoprotein subunit (mouse UniProt-code Q8K2B3). First, we will outline the in silico assay design including the choice of peptides based on a range of properties. We will further delineate different quantification strategies and introduce the reader to LC-MS assay development including the selection of the optimal peptide charge state and fragment ions as well as a discussion of the dynamic range of detection. The chapter will close with an application from the area of mitochondrial biology related to the quantification of a set of proteins isolated from mouse liver mitochondria in a study on mitochondrial respiratory flux decline in aging mouse muscle.


Assuntos
Mitocôndrias , Proteômica , Animais , Cromatografia Líquida , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Peptídeos/química , Proteômica/instrumentação , Proteômica/métodos , Espectrometria de Massas em Tandem
12.
Adv Exp Med Biol ; 1158: 119-142, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452139

RESUMO

The mitochondrion is a vital organelle that performs diverse cellular functions. In this regard, the cell has evolved various mechanisms dedicated to the maintenance of the mitochondrial proteome. Among them, AAA+ ATPase-associated proteases (AAA+ proteases) such as the Lon protease (LonP1), ClpXP complex, and the membrane-bound i-AAA, m-AAA and paraplegin facilitate the clearance of misfolded mitochondrial proteins to prevent the accumulation of cytotoxic protein aggregates. Furthermore, these proteases have additional regulatory functions in multiple biological processes that include amino acid metabolism, mitochondria DNA transcription, metabolite and cofactor biosynthesis, maturation and turnover of specific respiratory and metabolic proteins, and modulation of apoptosis, among others. In cancer cells, the increase in intracellular ROS levels promotes tumorigenic phenotypes and increases the frequency of protein oxidation and misfolding, which is compensated by the increased expression of specific AAA+ proteases as part of the adaptation mechanism. The targeting of AAA+ proteases has led to the discovery and development of novel anti-cancer compounds. Here, we provide an overview of the molecular characteristics and functions of the major mitochondrial AAA+ proteases and summarize recent research efforts in the development of compounds that target these proteases.


Assuntos
Proteínas Mitocondriais , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Ativação Enzimática , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/enzimologia , Neoplasias/fisiopatologia , Neoplasias/terapia , Protease La/metabolismo
13.
Adv Exp Med Biol ; 1158: 143-182, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452140

RESUMO

Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondrial lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre-analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed.


Assuntos
Biologia Computacional , Diabetes Mellitus Tipo 2 , Fígado , Mitocôndrias , Músculo Esquelético , Proteômica , Diabetes Mellitus Tipo 2/fisiopatologia , Humanos , Metabolismo dos Lipídeos , Fígado/fisiopatologia , Mitocôndrias/metabolismo , Músculo Esquelético/fisiopatologia
14.
Adv Exp Med Biol ; 1158: 197-216, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452142

RESUMO

Mitochondria are dynamic organelles that perform a number of interconnected tasks that are elegantly intertwined with the regulation of cell functions. This includes the provision of ATP, reactive oxygen species (ROS), and building blocks for the biosynthesis of macromolecules while also serving as signaling platforms for the cell. Although the functions executed by mitochondria are complex, at its core these roles are, to a certain degree, fulfilled by electron transfer reactions and the establishment of a protonmotive force (PMF). Indeed, mitochondria are energy conserving organelles that extract electrons from nutrients to establish a PMF, which is then used to drive ATP and NADPH production, solute import, and many other functions including the propagation of cell signals. These same electrons extracted from nutrients are also used to produce ROS, pro-oxidants that can have potentially damaging effects at high levels, but also serve as secondary messengers at low amounts. Mitochondria are also enriched with antioxidant defenses, which are required to buffer cellular ROS. These same redox buffering networks also fulfill another important role; regulation of proteins through the reversible oxidation of cysteine switches. The modification of cysteine switches with the antioxidant glutathione, a process called protein S-glutathionylation, has been found to play an integral role in controlling various mitochondrial functions. In addition, recent findings have demonstrated that disrupting mitochondrial protein S-glutathionylation reactions can have some dire pathological consequences. Accordingly, this chapter focuses on the role of mitochondrial cysteine switches in the modulation of different physiological functions and how defects in these pathways contribute to the development of disease.


Assuntos
Cisteína , Metabolismo Energético , Mitocôndrias , Espécies Reativas de Oxigênio , Animais , Cisteína/metabolismo , Humanos , Mitocôndrias/metabolismo , Oxirredução
15.
Adv Exp Med Biol ; 1173: 125-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456208

RESUMO

Friedreich's ataxia (FRDA) is a degenerative disease that affects both the central and the peripheral nervous systems and non-neural tissues including, mainly, heart, and endocrine pancreas. It is an autosomal recessive disease caused by a GAA triplet-repeat localized within an Alu sequence element in intron 1 of frataxin (FXN) gene, which encodes a mitochondrial protein FXN. This protein is essential for mitochondrial function by the involvement of iron-sulfur cluster biogenesis. The effects of its deficiency also include disruption of cellular, particularly mitochondrial, iron homeostasis, i.e., relatively more iron accumulated in mitochondria and less iron presented in cytosol. Though iron toxicity is commonly thought to be mediated via Fenton reaction, oxidative stress seems not to be the main problem to result in detrimental effects on cell survival, particularly neuron survival. Therefore, the basic research on FXN function is urgently demanded to understand the disease. This chapter focuses on the outcome of FXN expression, regulation, and function in cellular or animal models of FRDA and on iron pathophysiology in the affected tissues. Finally, therapeutic strategies based on the control of iron toxicity and iron cellular redistribution are considered. The combination of multiple therapeutic targets including iron, oxidative stress, mitochondrial function, and FXN regulation is also proposed.


Assuntos
Ataxia de Friedreich/fisiopatologia , Ferro/metabolismo , Animais , Humanos , Proteínas de Ligação ao Ferro/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Estresse Oxidativo
16.
Chem Biol Interact ; 311: 108789, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31401089

RESUMO

The cytotoxicity of a dinuclear imine-copper (II) complex 2, and its analogous mononuclear complex 1, toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2, a tyrosinase mimic, showed much higher activity in comparison to complex 1, and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1, both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2, while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2, in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity.


Assuntos
Complexos de Coordenação/química , Cobre/química , Iminas/química , Monofenol Mono-Oxigenase/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Humanos , Melaninas/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tubulina (Proteína)/metabolismo , Raios Ultravioleta
17.
Int J Nanomedicine ; 14: 5033-5050, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371945

RESUMO

Background: Repairs to deep skin wounds continue to be a difficult issue in clinical practice. A promising approach is to fabricate full-thickness skin substitutes with functions closely similar to those of the natural tissue. For many years, a three-dimensional (3D) collagen hydrogel has been considered to provide a physiological 3D environment for co-cultivation of skin fibroblasts and keratinocytes. This collagen hydrogel is frequently used for fabricating tissue-engineered skin analogues with fibroblasts embedded inside the hydrogel and keratinocytes cultivated on its surface. Despite its unique biological properties, the collagen hydrogel has insufficient stiffness, with a tendency to collapse under the traction forces generated by the embedded cells. Methods: The aim of our study was to develop a two-layer skin construct consisting of a collagen hydrogel reinforced by a nanofibrous poly-L-lactide (PLLA) membrane pre-seeded with fibroblasts. The attractiveness of the membrane for dermal fibroblasts was enhanced by coating it with a thin nanofibrous fibrin mesh. Results: The fibrin mesh promoted the adhesion, proliferation and migration of the fibroblasts upwards into the collagen hydrogel. Moreover, the fibroblasts spontaneously migrating into the collagen hydrogel showed a lower tendency to contract and shrink the hydrogel by their traction forces. The surface of the collagen was seeded with human dermal keratinocytes. The keratinocytes were able to form a basal layer of highly mitotically-active cells, and a suprabasal layer. Conclusion: The two-layer skin construct based on collagen hydrogel with spontaneously immigrated fibroblasts and reinforced by a fibrin-coated nanofibrous membrane seems to be promising for the construction of full-thickness skin substitute.


Assuntos
Colágeno/farmacologia , Fibrina/farmacologia , Hidrogéis/farmacologia , Membranas Artificiais , Nanofibras/química , Poliésteres/farmacologia , Pele Artificial , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Derme/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos
18.
Chem Commun (Camb) ; 55(72): 10740-10743, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31432813

RESUMO

We constructed a two-photon fluorescence ratio probe (CST) for in situ quantitative real-time detection of mitochondrial O2˙-. Fluorescence imaging showed that O2˙- was over-generated from mitochondria and conveyed to the cytoplasm via voltage-dependent anion channels in hepatic ischemia-reperfusion mice, damaging the important functional protein aconitase in the cytoplasm.


Assuntos
Fígado/metabolismo , Mitocôndrias/química , Imagem Óptica , Fótons , Traumatismo por Reperfusão/metabolismo , Superóxidos/química , Animais , Ânions/química , Ânions/metabolismo , Transporte Biológico , Corantes Fluorescentes/química , Camundongos , Mitocôndrias/metabolismo , Estrutura Molecular , Superóxidos/metabolismo
19.
Int J Nanomedicine ; 14: 4991-5015, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31371943

RESUMO

Purpose: This study evaluates the cytotoxicity of AuNPs coated with polyallylamine (AuNPs-PAA) and conjugated or not to the epidermal growth factor receptor (EGFR)-targeting antibody Cetuximab (AuNPs-PAA-Ctxb) in normal human kidney (HK-2), liver (THLE-2) and microvascular endothelial (TIME) cells, and compares it with two cancer cell lines that are EGFR-overexpressing (A431) or EGFR-negative (MDA-MB-453). Results: Conjugation of Cetuximab to AuNPs-PAA increased the AuNPs-PAA-Ctxb interactions with cells, but reduced their cytotoxicity. TIME cells exhibited the strongest reduction in viability after exposure to AuNPs-PAA(±Ctxb), followed by THLE-2, MDA-MB-453, HK-2 and A431 cells. This cell type-dependent sensitivity was strongly correlated to the inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR), and to the depolarization of the mitochondrial membrane potential. Both are suggested to initiate apoptosis, which was indeed detected in a concentration- and time-dependent manner. The role of oxidative stress in AuNPs-PAA(±Ctxb)-induced cytotoxicity was demonstrated by co-incubation of the cells with N-acetyl L-cysteine (NAC), which significantly decreased apoptosis and mitochondrial membrane depolarization. Conclusion: This study helps to identify the cells and tissues that could be sensitive to AuNPs and deepens the understanding of the risks associated with the use of AuNPs in vivo.


Assuntos
Antioxidantes/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab/farmacologia , Endocitose/efeitos dos fármacos , Glutationa Redutase/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Tamanho da Partícula , Poliaminas/química , Substâncias Protetoras/farmacologia , Eletricidade Estática , Tiorredoxina Dissulfeto Redutase/metabolismo
20.
Chem Commun (Camb) ; 55(67): 10015-10018, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31378791

RESUMO

We report the first neutral and water-soluble polymer capable of strong mitochondrial targeting in vitro and in vivo, zwitterionic poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate] (OPDEA). OPDEA is quickly internalized via macropinocytosis by various cancer cells and transferred into the mitochondria, which slightly lowers the mitochondrial membrane potential as determined by the JC-1 assay.


Assuntos
Mitocôndrias/metabolismo , Ácidos Polimetacrílicos/química , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Potencial da Membrana Mitocondrial , Camundongos , Imagem Óptica/métodos , Permeabilidade , Ácidos Polimetacrílicos/metabolismo , Solubilidade , Água
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA