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1.
Curr Opin Clin Nutr Metab Care ; 22(5): 355-362, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31145123

RESUMO

PURPOSE OF REVIEW: In addition to the currently available lysosomotropic drugs and autophagy whole-body knockout mouse models, we provide alternative methods that enable the modulation and detection of autophagic flux in vivo, discussing advantages and disadvantages of each method. RECENT FINDINGS: With the autophagosome-lysosome fusion inhibitor colchicine in skeletal muscle and temporal downregulation of autophagy using a novel Autophagy related 5-short hairpin RNA (Atg5-shRNA) mouse model we mention two models that directly modulate autophagy flux in vivo. Furthermore, methods to quantify autophagy flux, such as mitophagy transgenic reporters, in situ immunofluorescent staining and multispectral imaging flow cytometry, in mature skeletal muscle and cells are addressed. SUMMARY: To achieve clinical benefit, less toxic, temporary and cell-type-specific modulation of autophagy should be pursued further. A temporary knockdown as described for the Atg5-shRNA mice could provide a first insight into possible implications of autophagy inhibition. However, it is also important to take a closer look into the methods to evaluate autophagy after harvesting the tissue. In particular caution is required when experimental conditions can influence the final measurement and this should be pretested carefully.

2.
Cardiovasc Ultrasound ; 17(1): 7, 2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31010431

RESUMO

Echocardiography is the most commonly applied technique for non-invasive assessment of cardiac function in small animals. Manual tracing of endocardial borders is time consuming and varies with operator experience. Therefore, we aimed to evaluate a novel automated two-dimensional software algorithm (Auto2DE) for small animals and compare it to the standard use of manual 2D-echocardiographic assessment (2DE). We hypothesized that novel Auto2DE will provide rapid and robust data sets, which are in agreement with manually assessed data of animals.2DE and Auto2DE were carried out using a high-resolution imaging-system for small animals. First, validation cohorts of mouse and rat cine loops were used to compare Auto2DE against 2DE. These data were stratified for image quality by a blinded expert in small animal imaging. Second, we evaluated 2DE and Auto2DE in four mouse models and four rat models with different cardiac pathologies.Automated assessment of LV function by 2DE was faster than conventional 2DE analysis and independent of operator experience levels. The accuracy of Auto2DE-assessed data in healthy mice was dependent on cine loop quality, with excellent agreement between Auto2DE and 2DE in cine loops with adequate quality. Auto2DE allowed for valid detection of impaired cardiac function in animal models with pronounced cardiac phenotypes, but yielded poor performance in diabetic animal models independent of image quality.Auto2DE represents a novel automated analysis tool for rapid assessment of LV function, which is suitable for data acquisition in studies with good and very good echocardiographic image quality, but presents systematic problems in specific pathologies.

3.
Nutrients ; 11(3)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866427

RESUMO

For centuries, Amaranthus sp. were used as food, ornamentals, and medication. Molecular mechanisms, explaining the health beneficial properties of amaranth, are not yet understood, but have been attributed to secondary metabolites, such as phenolic compounds. One of the most abundant phenolic compounds in amaranth leaves is 2-caffeoylisocitric acid (C-IA) and regarding food occurrence, C-IA is exclusively found in various amaranth species. In the present study, the anti-inflammatory activity of C-IA, chlorogenic acid, and caffeic acid in LPS-challenged macrophages (RAW 264.7) has been investigated and cellular contents of the caffeic acid derivatives (CADs) were quantified in the cells and media. The CADs were quantified in the cell lysates in nanomolar concentrations, indicating a cellular uptake. Treatment of LPS-challenged RAW 264.7 cells with 10 µM of CADs counteracted the LPS effects and led to significantly lower mRNA and protein levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin 6, by directly decreasing the translocation of the nuclear factor κB/Rel-like containing protein 65 into the nucleus. This work provides new insights into the molecular mechanisms that attribute to amaranth's anti-inflammatory properties and highlights C-IA's potential as a health-beneficial compound for future research.


Assuntos
Amaranthus/química , Anti-Inflamatórios/farmacologia , Ácidos Cafeicos/farmacologia , Isocitratos/farmacologia , NF-kappa B/metabolismo , Animais , Ácidos Cafeicos/química , Citocinas/metabolismo , Isocitratos/química , Lipopolissacarídeos/efeitos adversos , Camundongos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
4.
Cardiovasc Ultrasound ; 16(1): 10, 2018 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-29966517

RESUMO

BACKGROUND: The assessment of ventricular volumes using conventional echocardiography methods is limited with regards to the need of geometrical assumptions. In the present study, we aimed to evaluate a novel commercial system for three-dimensional echocardiography (3DE) in preclinical models by direct comparison with conventional 1D- and 2D-echocardiography (1DE; 2DE) and the gold-standard technique magnetic resonance imaging (MRI). Further, we provide a standard operating protocol for image acquisition and analysis with 3DE. METHODS: 3DE was carried out using a 30 MHz center frequency transducer coupled to a Vevo®3100 Imaging System. We evaluated under different experimental conditions: 1) in vitro phantom measurements served as controlled setting in which boundaries were clearly delineated; 2) a validation cohort composed of healthy C57BL/6 J mice and New Zealand Obese (NZO) mice was used in order to validate 3DE against cardiac MRI; 3) a standard mouse model of pressure overload induced-heart failure was investigated to estimate the value of 3DE. RESULTS: First, in vitro volumetry revealed good agreement between 3DE assessed volumes and the MRI-assessed volumes. Second, cardiac volume determination with 3DE showed smaller mean differences compared to cardiac MRI than conventional 1DE and 2DE. Third, 3DE was suitable to detect reduced ejection fractions in heart failure mice. Fourth, inter- and intra-observer variability of 3DE showed good to excellent agreement regarding absolute volumes in healthy mice, whereas agreement rates for the relative metrics ejection fraction and stroke volume demonstrated good to moderate observer variabilities. CONCLUSIONS: 3DE provides a novel method for accurate volumetry in small animals without the need for spatial assumptions, demonstrating a technique for an improved analysis of ventricular function. Further validation work and highly standardized image analyses are required to increase reproducibility of this approach.


Assuntos
Ecocardiografia Tridimensional , Insuficiência Cardíaca/diagnóstico por imagem , Volume Sistólico , Animais , Modelos Animais de Doenças , Ecocardiografia , Interpretação de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador , Imagem por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Variações Dependentes do Observador , Reprodutibilidade dos Testes
5.
Mech Ageing Dev ; 170: 72-81, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28755850

RESUMO

Aging is accompanied by the accumulation of cellular damage over time in response to stress, lifestyle and environmental factors ultimately leading to age-related diseases and death. Additionally, the number of senescent cells increases with age. Senescence is most likely not a static endpoint, it represents a series of hallmarks including morphological changes, alterations in protein turnover and accumulation of protein aggregates. The importance of protein oxidation and aggregate accumulation in the progression of aging is not yet fully understood and research to what extent the accumulation of oxidized proteins has an effect on senescence and the aging process is still ongoing. To study the mechanisms of aging, the impact of senescence and the role of protein aggregates on the aging process, cell culture models are useful tools. Most notably stress induced premature senescence (SIPS) models have contributed to the identification of mechanisms involved in the aging process and helped unravel the age-related changes in proteolysis and the importance of protein aggregation. Here we review characteristics of replicative and premature senescence, how to induce most frequently used senescence models and gained knowledge on age-related changes in the major proteolytic systems.


Assuntos
Envelhecimento/metabolismo , Senescência Celular , Modelos Biológicos , Agregação Patológica de Proteínas/metabolismo , Proteólise , Envelhecimento/patologia , Animais , Humanos , Agregação Patológica de Proteínas/patologia
6.
Redox Biol ; 11: 673-681, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28160744

RESUMO

Mitochondria have been in the focus of oxidative stress and aging research for decades due to their permanent production of ROS during the oxidative phosphorylation. The hypothesis exists that mitochondria are involved in the formation of lipofuscin, an autofluorescent protein aggregate that accumulates progressively over time in lysosomes of post-mitotic and senescent cells. To investigate the influence and involvement of mitochondria in lipofuscinogenesis, we analyzed lipofuscin amounts as well as the mitochondrial function in young and senescent cells. In addition we used an aging model and Lon protease deficient HeLa cells to investigate the influence of mitochondrial degradation processes on lipofuscin formation. We were able to show that mitophagy is impaired in senescent cells resulting in an increased mitochondrial mass and superoxide formation. In addition, the inhibition of mitochondrial fission leads to increased lipofuscin formation. Moreover, we observed that Lon protease downregulation is linked to a higher lipofuscinogenesis whereas the application of the mitochondrial-targeted antioxidant mitoTEMPO is able to prevent the accumulation of this protein aggregate.


Assuntos
Envelhecimento/metabolismo , Senescência Celular/genética , Lipofuscina/biossíntese , Mitocôndrias/metabolismo , Protease La/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Animais , Autofagia/genética , Células HeLa , Humanos , Lipofuscina/metabolismo , Lisossomos/metabolismo , Degradação Mitocondrial/genética , Estresse Oxidativo/genética , Agregados Proteicos/genética , Espécies Reativas de Oxigênio/metabolismo
7.
Redox Biol ; 11: 482-501, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28086196

RESUMO

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset.


Assuntos
Envelhecimento/genética , Antioxidantes/metabolismo , Senescência Celular/genética , Estresse Oxidativo/genética , Envelhecimento/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oxidantes/metabolismo
8.
Redox Biol ; 10: 266-273, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27825071

RESUMO

The overall decrease in proteolytic activity in aging can promote and accelerate protein accumulation and metabolic disturbances. To specifically analyze changes in macroautophagy (MA) we quantified different autophagy-related proteins (ATGs) in young, adult and old murine tissue as well as in young and senescent human fibroblasts. Thus, we revealed significantly reduced levels of ATG5-ATG12, LC3-II/LC3-I ratio, Beclin-1 and p62 in old brain tissue and senescent human fibroblasts. To investigate the role of mTOR, the protein itself and its target proteins p70S6 kinase and 4E-BP1 were quantified. Significant increased mTOR protein levels were determined in old tissue and cells. Determination of phosphorylated and basal amount of both proteins suggested higher mTOR activity in old murine tissue and senescent human fibroblasts. Besides the reduced levels of ATGs, mTOR can additionally reduce MA, promoting further acceleration of protein accumulation and metabolic disturbances during aging.


Assuntos
Envelhecimento/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Encéfalo/metabolismo , Fibroblastos/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Senescência Celular , Regulação para Baixo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo
9.
Free Radic Biol Med ; 101: 325-333, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27789294

RESUMO

Changes in the two main intracellular degradation systems, the Ubiquitin-Proteasome System and the Autophagy-Lysosome pathway (ALP) are widely discussed as a hallmark of the aging process. To follow the age-related behavior of both degradation systems we examined their impact on ferritin, known to be degradable by both. Ferritin H was analyzed in young and senescent human fibroblasts, revealing a higher steady-state level in the senescent cells. By blocking both proteolytic systems, we confirmed that particularly the ALP plays a crucial role in ferritin H turnover. However, an unexpected increase in lysosomal activity in the senescent cells, suggests a dysregulation in the autophagy pathway. To further investigate the impaired ferritin H turnover, confocal microscopic colocalization studies of ferritin H with lysosomal-associated membrane protein 2a (Lamp2a) and monodansylcadaverine (MDC) were performed and clearly revealed the degradation of ferritin by macroautophagy. By induction of autophagy via inhibition of mTOR using rapamycin an increase of ferritin H turnover was obtained in senescent cells, demonstrating a mTOR dependent reduction of autophagy in senescent human fibroblasts.


Assuntos
Apoferritinas/metabolismo , Autofagia/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Serina-Treonina Quinases TOR/genética , Autofagia/genética , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Senescência Celular/efeitos dos fármacos , Desferroxamina/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Prepúcio do Pênis/metabolismo , Expressão Gênica , Hemina/farmacologia , Humanos , Cinética , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Masculino , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
10.
Redox Biol ; 2: 411-29, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24624331

RESUMO

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.


Assuntos
Produtos Finais de Glicação Avançada/fisiologia , Inflamação/etiologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/fisiologia , Envelhecimento/fisiologia , Animais , Osso e Ossos/metabolismo , Proteínas na Dieta/efeitos adversos , Proteínas na Dieta/farmacocinética , Humanos , Hiperglicemia/metabolismo , Sistema Imunitário/metabolismo , Inflamação/metabolismo , Peroxidação de Lipídeos , Pulmão/metabolismo , Reação de Maillard , Modelos Biológicos , NF-kappa B/fisiologia , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Polímeros/metabolismo , Agregados Proteicos , Transporte Proteico , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Depuradores/fisiologia
11.
Curr Pharm Des ; 20(18): 3040-51, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24079764

RESUMO

A number of studies reported a relation between longevity, oxidative stress and age-related diseases. Every aerobic organism is inevitably exposed to a permanent flux of free radicals and oxidants. Due to the limited activity of antioxidant and repair mechanisms, levels of reactive oxygen species can increase during aging. Protein damage caused by elevated levels of free radicals or oxidants has an important influence on cellular viability and leads to malfunction of proteins in aged cells. In addition, modified and impaired proteins can cross-link and form the bases of many senescence-associated alterations and also of neurodegenerative diseases. To ensure the maintenance of normal cellular functions, eukaryotic cells exert proteolysis through two systems: the proteasomal system and the lysosomal system, which is degrading cellular components after autophagy. During cellular differentiation and aging, both systems are subject to extensive changes that significantly affect their proteolytic activity. It has been suggested that highly modified proteins and undegradable protein aggregates also affect the intracellular proteolytic systems. Therefore, it is essential to understand the relationship between protein oxidation, intracellular proteolytic systems and cellular defence mechanisms.


Assuntos
Envelhecimento/fisiologia , Proteínas/metabolismo , Proteólise , Animais , Antioxidantes/metabolismo , Autofagia/fisiologia , Radicais Livres/metabolismo , Humanos , Oxirredução , Estresse Oxidativo/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
Biochem J ; 448(1): 127-39, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22892029

RESUMO

AGEs (advanced glycation-end products) accumulate during aging and several pathologies such as Alzheimer's disease and diabetes. These protein products are known to inhibit proteolytic pathways. Moreover, AGEs are known to be involved in the activation of immune responses. In the present study we demonstrate that AGEs induce the expression of immunoproteasomal subunits. To elucidate a molecular basis underlying the observed effects we were able to demonstrate an activation of the Jak2 (Janus kinase 2)/STAT1 (signal transducer and activator of transcription 1) pathway. Inhibition of Jak2 by AG-490 and STAT1 by specific siRNA (small interfering RNA) abolished AGE-induced expression of immunoproteasomal subunits. Furthermore, silencing of RAGE (receptor for AGEs) revealed that AGE-induced up-regulation of the immunoproteasome is mediated by a RAGE signalling process. Thus we have described for the first time that the signalling pathway of Jak2 and STAT1 activated by AGEs via RAGE is involved in the induction of the immunoproteasome.


Assuntos
Produtos Finais de Glicação Avançada/farmacologia , Janus Quinase 2/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Imunológicos/fisiologia , Fator de Transcrição STAT1/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral/metabolismo , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica/genética , Interferon gama/fisiologia , Macrófagos/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/farmacologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Soroalbumina Bovina/farmacologia , Transcrição Genética/efeitos dos fármacos , Tirfostinas/farmacologia
13.
Free Radic Biol Med ; 53(4): 916-25, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22705366

RESUMO

Protein carbonylation is a common feature in cells exposed to oxidants, leading to protein dysfunction and protein aggregates. Actin, which is involved in manifold cellular processes, is a sensitive target protein to this oxidative modification. T-cell proteins have been widely described to be sensitive targets to oxidative modifications. The aim of this work was to test whether the formation of protein aggregates contributes to the impaired proliferation of Jurkat cells after oxidative stress and to test whether actin as a major oxidation-prone cytoskeletal protein is an integral part of such protein aggregates. We used Jurkat cells, an established T-cell model, showing the formation of actin aggregates along with the decrease of proteasome activity. The presence of these protein aggregates inhibits Jurkat proliferation even under conditions not influencing viability. As a conclusion, we propose that an oxidative environment leads to actin aggregates contributing to T-cell cellular functional impairment.


Assuntos
Actinas/metabolismo , Carbonilação Proteica , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Peróxido de Hidrogênio/farmacologia , Células Jurkat , Oxidantes/farmacologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Transporte Proteico , Linfócitos T/metabolismo , Linfócitos T/fisiologia
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