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  1 / 7526 MEDLINE  
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[PMID]:28456689
[Au] Autor:Mandic A; Strebinger D; Regali C; Phillips NE; Suter DM
[Ad] Endereço:The Institute of Bioengineering (IBI), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland.
[Ti] Título:A novel method for quantitative measurements of gene expression in single living cells.
[So] Source:Methods;120:65-75, 2017 May 01.
[Is] ISSN:1095-9130
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Gene expression is at the heart of virtually any biological process, and its deregulation is at the source of numerous pathological conditions. While impressive progress has been made in genome-wide measurements of mRNA and protein expression levels, it is still challenging to obtain highly quantitative measurements in single living cells. Here we describe a novel approach based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse microscopy. Using luminescence microscopy, fluctuations of protein expression levels can be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. The integrated protein decay reporter allows measuring protein degradation rates in the absence of protein synthesis inhibitors, and in combination with absolute protein levels allows determining absolute amounts of proteins synthesized over the cell cycle. Finally, the internal tag can be excised by inducible expression of Cre recombinase, which enables to estimate endogenous mRNA half-lives. Our method thus opens new avenues in quantitative analysis of gene expression in single living cells.
[Mh] Termos MeSH primário: Imagem Molecular/métodos
Proteínas/genética
Análise de Célula Única/métodos
Coloração e Rotulagem/métodos
Transcrição Genética
[Mh] Termos MeSH secundário: Animais
Linhagem Celular
Genes Reporter/genética
Vetores Genéticos/genética
Meia-Vida
Integrases/genética
Lentivirus/genética
Luminescência
Camundongos
Microscopia de Fluorescência/métodos
Imagem Molecular/instrumentação
Proteínas/química
Proteínas/metabolismo
Proteólise
RNA Mensageiro/genética
RNA Mensageiro/metabolismo
Análise de Célula Única/instrumentação
Coloração e Rotulagem/instrumentação
Imagem com Lapso de Tempo/instrumentação
Imagem com Lapso de Tempo/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Proteins); 0 (RNA, Messenger); EC 2.7.7.- (Cre recombinase); EC 2.7.7.- (Integrases)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180309
[Lr] Data última revisão:
180309
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170501
[St] Status:MEDLINE


  2 / 7526 MEDLINE  
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[PMID]:29180866
[Au] Autor:Lapin NA; Vergara LA; Mackeyev Y; Newton JM; Dilliard SA; Wilson LJ; Curley SA; Serda RE
[Ad] Endereço:Michael E DeBakey Department of Surgery, Baylor College of Medicine.
[Ti] Título:Biotransport kinetics and intratumoral biodistribution of malonodiserinolamide-derivatized [60]fullerene in a murine model of breast adenocarcinoma.
[So] Source:Int J Nanomedicine;12:8289-8307, 2017.
[Is] ISSN:1178-2013
[Cp] País de publicação:New Zealand
[La] Idioma:eng
[Ab] Resumo:[60]Fullerene is a highly versatile nanoparticle (NP) platform for drug delivery to sites of pathology owing to its small size and both ease and versatility of chemical functionalization, facilitating multisite drug conjugation, drug targeting, and modulation of its physicochemical properties. The prominent and well-characterized role of the enhanced permeation and retention (EPR) effect in facilitating NP delivery to tumors motivated us to explore vascular transport kinetics of a water-soluble [60]fullerene derivatives using intravital microscopy in an immune competent murine model of breast adenocarcinoma. Herein, we present a novel local and global image analysis of vascular transport kinetics at the level of individual tumor blood vessels on the micron scale and across whole images, respectively. Similar to larger nanomaterials, [60]fullerenes displayed rapid extravasation from tumor vasculature, distinct from that in normal microvasculature. Temporal heterogeneity in fullerene delivery to tumors was observed, demonstrating the issue of nonuniform delivery beyond spatial dimensions. Trends in local region analysis of fullerene biokinetics by fluorescence quantification were in agreement with global image analysis. Further analysis of intratumoral vascular clearance rates suggested a possible enhanced penetration and retention effect of the fullerene compared to a 70 kDa vascular tracer. Overall, this study demonstrates the feasibility of tracking and quantifying the delivery kinetics and intratumoral biodistribution of fullerene-based drug delivery platforms, consistent with the EPR effect on short timescales and passive transport to tumors.
[Mh] Termos MeSH primário: Adenocarcinoma/tratamento farmacológico
Sistemas de Liberação de Medicamentos/métodos
Fulerenos/farmacocinética
Neoplasias Mamárias Experimentais/tratamento farmacológico
Nanopartículas/química
[Mh] Termos MeSH secundário: Animais
Difusão Dinâmica da Luz
Feminino
Fluorescência
Fulerenos/química
Microscopia Intravital/métodos
Cinética
Camundongos Endogâmicos BALB C
Microscopia Eletrônica de Varredura
Imagem Molecular/métodos
Solubilidade
Distribuição Tecidual
Água/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Fullerenes); 059QF0KO0R (Water)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180305
[Lr] Data última revisão:
180305
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171129
[St] Status:MEDLINE
[do] DOI:10.2147/IJN.S138641


  3 / 7526 MEDLINE  
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[PMID]:29367528
[Au] Autor:Oshima S
[Ad] Endereço:Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU).
[Ti] Título:[Autoimmune diseases and ubiquitin system].
[So] Source:Nihon Rinsho Meneki Gakkai Kaishi;40(6):442-449, 2017.
[Is] ISSN:1349-7413
[Cp] País de publicação:Japan
[La] Idioma:jpn
[Ab] Resumo:  Cytokines play important roles in the pathogenesis of autoimmune diseases. Anti-TNFα antibody therapy for rheumatoid arthritis, Crohn's disease, and psoriasis has made enough progress to change its treatment goal. This review focuses on the recent advances that have been made in understanding TNFR signaling through ubiquitin system. Genome-wide association studies (GWAS) identified numerous susceptibility loci associated with autoimmune diseases. Ubiquitin related genes TNFAIP3 and TNIP1 have been linked to multiple autoimmune diseases. Here, we review the importance of TNFAIP3 and TNIP1-mediated regulation of ubiquitin-dependent signaling. To monitor the dynamics of ubiquitin chain formation in vivo, we have developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay. The PolyUb-FC assay has the advantage that monoubiquitination is non-fluorescent and chain-specific poly-ubiquitination can be directly visualized in living cells without using antibodies. The PolyUb-FC will be a useful tool for analyzing the dynamics of polyubiquitin chain generation.
[Mh] Termos MeSH primário: Doenças Autoimunes/etiologia
Ubiquitina/metabolismo
[Mh] Termos MeSH secundário: Animais
Anticorpos Monoclonais/uso terapêutico
Doenças Autoimunes/tratamento farmacológico
Doenças Autoimunes/genética
Proteínas de Ligação a DNA/fisiologia
Predisposição Genética para Doença
Estudo de Associação Genômica Ampla
Seres Humanos
Camundongos
Imagem Molecular/métodos
Transdução de Sinais/genética
Proteína 3 Induzida por Fator de Necrose Tumoral alfa/fisiologia
Fator de Necrose Tumoral alfa/imunologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Antibodies, Monoclonal); 0 (DNA-Binding Proteins); 0 (TNIP1 protein, human); 0 (Tumor Necrosis Factor-alpha); 0 (Ubiquitin); EC 3.4.19.12 (TNFAIP3 protein, human); EC 3.4.19.12 (Tumor Necrosis Factor alpha-Induced Protein 3)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180126
[St] Status:MEDLINE
[do] DOI:10.2177/jsci.40.442


  4 / 7526 MEDLINE  
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[PMID]:29239411
[Au] Autor:Ogasawara H; Grzybowski M; Hosokawa R; Sato Y; Taki M; Yamaguchi S
[Ad] Endereço:Department of Chemistry, Graduate School of Science and Integrated Research Consortium on Chemical Sciences (IRCCS), Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan.
[Ti] Título:A far-red fluorescent probe based on a phospha-fluorescein scaffold for cytosolic calcium imaging.
[So] Source:Chem Commun (Camb);54(3):299-302, 2018 Jan 02.
[Is] ISSN:1364-548X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:The far-red emissive fluorescent probe CaPF-1 based on a phospha-fluorescein scaffold enables the detection of cytosolic calcium ions in living cells. The probe can be excited in the red region (λ = 636 nm) and exhibits a sufficiently high fluorescence turn-on response in the far-red region (λ = 663 nm) upon complexation with calcium ions. The hydrophilic and anionic characteristics of this phospha-fluorescein fluorophore allowed the cytosolic localization of CaPF-1. Moreover, it was possible to visualize histamine-induced calcium oscillation in HeLa cells using CaPF-1.
[Mh] Termos MeSH primário: Cálcio/análise
Óxidos P-Cíclicos/farmacologia
Fluoresceínas/farmacologia
Corantes Fluorescentes/farmacologia
[Mh] Termos MeSH secundário: Cálcio/metabolismo
Óxidos P-Cíclicos/síntese química
Óxidos P-Cíclicos/química
Fluoresceínas/síntese química
Fluoresceínas/química
Fluorescência
Corantes Fluorescentes/síntese química
Corantes Fluorescentes/química
Células HeLa
Histamina/metabolismo
Seres Humanos
Concentração de Íons de Hidrogênio
Microscopia Confocal
Imagem Molecular
Imagem Óptica
Receptores Histamínicos H1/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Cyclic P-Oxides); 0 (Fluoresceins); 0 (Fluorescent Dyes); 0 (Receptors, Histamine H1); 820484N8I3 (Histamine); SY7Q814VUP (Calcium)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171215
[St] Status:MEDLINE
[do] DOI:10.1039/c7cc07344e


  5 / 7526 MEDLINE  
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[PMID]:29372686
[Au] Autor:Hnilicová P; Richterová R; Kantorová E; Bittsanský M; Baranovicová E; Dobrota D
[Ad] Endereço:Division of Neurosciences at Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia. dusan.dobrota@jfmed.uniba.sk.
[Ti] Título:Proton MR spectroscopic imaging of human glioblastomas at 1.5 Tesla.
[So] Source:Gen Physiol Biophys;36(5):531-537, 2017 Dec.
[Is] ISSN:0231-5882
[Cp] País de publicação:Slovakia
[La] Idioma:eng
[Ab] Resumo:In this study we evaluated clinical feasibility of proton magnetic resonance spectroscopy metabolite mapping (1H MRSI) by using 1.5 Tesla MR-scanner in 10 patients with high-grade glioblastoma. In vivo 1H MRSI performed with a relatively short scan time of 20 minutes enabled to obtain comprehensive information about metabolic changes in glioblastoma and adjacent tissues namely in the peritumoral edema, in the middle and solid part of the tumor, and in the normal-appearing brain tissue. Spectroscopically it was possible to identify initiation of neuronal cell death in the solid tumorous tissue via decreased N-acetyl-aspartate to creatine ratio (↓ tNAA/tCr) and expanding carcinogenesis reflected in elevated choline ratios (↑ tCho/tCr and tCho/tNAA). We showed also the central necrosis of glioblastoma accompanied by the tissue hypoxia, which were apparent as increased lactate and lipids ratios (↑ Lac/tCr and lip/Lac). Metabolic changes were noticeable also in the peritumoral area, showing the glioblastoma infiltration into the surrounding tissues. In intracranial tumors, 1H MRSI performed on 1.5 Tesla field strength was sufficient to provide information about the stage of carcinogenesis, tumor expansion or necrotization and thus it could be considered as a useful diagnostic tool in oncology.
[Mh] Termos MeSH primário: Biomarcadores Tumorais/análise
Neoplasias Encefálicas/química
Neoplasias Encefálicas/diagnóstico
Glioblastoma/química
Glioblastoma/diagnóstico
Espectroscopia de Prótons por Ressonância Magnética/métodos
[Mh] Termos MeSH secundário: Estudos de Viabilidade
Feminino
Seres Humanos
Imagem por Ressonância Magnética/métodos
Masculino
Meia-Idade
Imagem Molecular/métodos
Reprodutibilidade dos Testes
Sensibilidade e Especificidade
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Biomarkers, Tumor)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180223
[Lr] Data última revisão:
180223
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180127
[St] Status:MEDLINE
[do] DOI:10.4149/gpb_2017027


  6 / 7526 MEDLINE  
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[PMID]:29199986
[Au] Autor:Tarver CL; Pusey M
[Ad] Endereço:Department of Biological Science, University of Alabama in Huntsville, Huntsville, AL 35899, USA.
[Ti] Título:A low-cost method for visible fluorescence imaging.
[So] Source:Acta Crystallogr F Struct Biol Commun;73(Pt 12):657-663, 2017 Dec 01.
[Is] ISSN:2053-230X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins ß-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using ß-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.
[Mh] Termos MeSH primário: Corantes Fluorescentes/química
Imagem Molecular/instrumentação
Imagem Molecular/métodos
Proteínas/química
[Mh] Termos MeSH secundário: Cor
Concanavalina A/química
Custos e Análise de Custo
Cristalização
Desenho de Equipamento
Fluorescência
Lactoglobulinas/química
Imagem Molecular/economia
Rodaminas/química
Smartphone/economia
Smartphone/instrumentação
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (6-carboxyrhodamine-6G); 0 (Fluorescent Dyes); 0 (Lactoglobulins); 0 (Proteins); 0 (Rhodamines); 11028-71-0 (Concanavalin A)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180222
[Lr] Data última revisão:
180222
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171205
[St] Status:MEDLINE
[do] DOI:10.1107/S2053230X17015941


  7 / 7526 MEDLINE  
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[PMID]:29273416
[Au] Autor:Kimura H; Ogawa Y; Fujimoto H; Mukai E; Kawashima H; Arimitsu K; Toyoda K; Fujita N; Yagi Y; Hamamatsu K; Murakami T; Murakami A; Ono M; Nakamoto Y; Togashi K; Inagaki N; Saji H
[Ad] Endereço:Department of Patho-Functional Bioanalysis, Kyoto University Graduate School of Pharmaceutical Sciences, 46-29, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan; Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyot
[Ti] Título:Evaluation of F-labeled exendin(9-39) derivatives targeting glucagon-like peptide-1 receptor for pancreatic ß-cell imaging.
[So] Source:Bioorg Med Chem;26(2):463-469, 2018 01 15.
[Is] ISSN:1464-3391
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:ß-cell mass (BCM) is known to be decreased in subjects with type-2 diabetes (T2D). Quantitative analysis for BCM would be useful for understanding how T2D progresses and how BCM affects treatment efficacy and for earlier diagnosis of T2D and development of new therapeutic strategies. However, a noninvasive method to measure BCM has not yet been developed. We developed four F-labeled exendin(9-39) derivatives for ß-cell imaging by PET: [ F]FB9-Ex(9-39), [ F]FB12-Ex(9-39), [ F]FB27-Ex(9-39), and [ F]FB40-Ex(9-39). Affinity to the glucagon-like peptide-1 receptor (GLP-1R) was evaluated with dispersed islet cells of ddY mice. Uptake of exendin(9-39) derivatives in the pancreas as well as in other organs was evaluated by a biodistribution study. Small-animal PET study was performed after injecting [ F]FB40-Ex(9-39). FB40-Ex(9-39) showed moderate affinity to the GLP-1R. Among all of the derivatives, [ F]FB40-Ex(9-39) resulted in the highest uptake of radioactivity in the pancreas 30 min after injection. Moreover, it showed significantly less radioactivity accumulated in the liver and kidney, resulting in an overall increase in the pancreas-to-organ ratio. In the PET imaging study, pancreas was visualized at 30 min after injection of [ F]FB40-Ex(9-39). [ F]FB40-Ex(9-39) met the basic requirements for an imaging probe for GLP-1R in pancreatic ß-cells. Further enhancement of pancreatic uptake and specific binding to GLP-1R will lead to a clear visualization of pancreatic ß-cells.
[Mh] Termos MeSH primário: Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo
Ilhotas Pancreáticas/metabolismo
Imagem Molecular
Fragmentos de Peptídeos/farmacocinética
Compostos Radiofarmacêuticos/farmacocinética
[Mh] Termos MeSH secundário: Animais
Relação Dose-Resposta a Droga
Radioisótopos de Flúor
Ilhotas Pancreáticas/citologia
Masculino
Camundongos
Camundongos Endogâmicos
Estrutura Molecular
Fragmentos de Peptídeos/administração & dosagem
Fragmentos de Peptídeos/química
Tomografia por Emissão de Pósitrons
Compostos Radiofarmacêuticos/administração & dosagem
Compostos Radiofarmacêuticos/química
Relação Estrutura-Atividade
Distribuição Tecidual
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Fluorine Radioisotopes); 0 (Glucagon-Like Peptide-1 Receptor); 0 (Peptide Fragments); 0 (Radiopharmaceuticals); 5313W10MYT (exendin (9-39))
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180220
[Lr] Data última revisão:
180220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171224
[St] Status:MEDLINE


  8 / 7526 MEDLINE  
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[PMID]:28470526
[Au] Autor:Friedrich O; Head SI
[Ad] Endereço:Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Institute of Medical Biotechnology, Paul-Gordan-Street 3, Erlangen, 91052, Germany. oliver.friedrich@fau.de.
[Ti] Título:Quantitative Ratiometric Ca Imaging to Assess Cell Viability.
[So] Source:Methods Mol Biol;1601:171-193, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Viability of cells is strongly related to their Ca homeostasis. Ca signal fluctuations can be on a slow time scale, e.g., in non-excitable cells, but also in the range of tens of milliseconds for excitable cells, such as nerve and muscle. Muscle fibers respond to electrical stimulation with Ca transients that exceed their resting basal level about 100 times. Fluorescent Ca dyes have become an indispensable means to monitor Ca fluctuations in living cells online. Fluorescence intensity of such "environmental dyes" relies on a buffer-ligand interaction which is not only governed by laws of mass action but also by binding and unbinding kinetics that have to be considered for proper Ca kinetics and amplitude validation. The concept of Ca dyes including the different approaches using ratiometric and non-ratiometric dyes, the way to correctly choose dyes according to their low-/high-affinity properties and kinetics as well as staining techniques, and in situ calibration are reviewed and explained. We provide detailed protocols to apply ratiometric Fura-2 imaging of resting Ca and Ca fluctuations during field-stimulation in single isolated skeletal muscle cells and how to translate fluorescence intensities into absolute Ca concentrations using appropriate calibration techniques.
[Mh] Termos MeSH primário: Sinalização do Cálcio
Cálcio/metabolismo
Sobrevivência Celular
Imagem Molecular/métodos
[Mh] Termos MeSH secundário: Animais
Cálcio/análise
Quelantes de Cálcio/química
Calibragem
Estimulação Elétrica
Corantes Fluorescentes/química
Fura-2/química
Cinética
Microscopia de Fluorescência
Fibras Musculares Esqueléticas/citologia
Imagem Óptica/métodos
Análise de Célula Única
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Calcium Chelating Agents); 0 (Fluorescent Dyes); SY7Q814VUP (Calcium); TSN3DL106G (Fura-2)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180219
[Lr] Data última revisão:
180219
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170505
[St] Status:MEDLINE
[do] DOI:10.1007/978-1-4939-6960-9_14


  9 / 7526 MEDLINE  
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[PMID]:29232953
[Au] Autor:Wang L; Zhang Y; Wang D; Wang M; Wang Y; Feng J
[Ad] Endereço:Research and Development Center of Biorational Pesticide, Northwest A&F University , Yangling 712100, Shaanxi, China.
[Ti] Título:Mitochondrial Signs and Subcellular Imaging Provide Insight into the Antifungal Mechanism of Carabrone against Gaeumannomyces graminis var. tritici.
[So] Source:J Agric Food Chem;66(1):81-90, 2018 Jan 10.
[Is] ISSN:1520-5118
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Carabrone, a botanical bicyclic sesquiterpenic lactone, has broad-spectrum antifungal activity and is particularly efficient against the devastating phytopathogen Gaeumannomyces graminis var. tritici (Ggt). The antifungal mechanism of carabrone against Ggt, however, remains unclear. The main objective of this study was to investigate the subcellular localization of carabrone in Ggt to gain a better understanding of its mechanism of action. When Ggt was exposed to carabrone (EC value of 28.45 µg/mL) for 7 days, a decline in mitochondrial concentration together with some obvious alternations in mitochondrial structure, including hazy outlines, medullary transitions, excess accumulation of unclear settlings, and vacuolar degeneration, were observed, indicating that carbrone may act on the mitochondria directly. A fluorescent conjugate (TTY) was thus designed and synthesized as a surrogate of carabrone that possessed comparable antifungal activity against Ggt (EC of 33.68 µg/mL). Additionally, a polyclonal antibody specific to carabrone and with a high titer (256 000) was also prepared by immunizing mice. Subsequently, two imaging techniques, the use of the fluorescent conjugate (FC) and immunofluorescence (IF), were applied to determine the subcellular localization of carabrone. Both FC and IF fluorescent signals demonstrated its mitochondrial localization with a Pearson's coefficient of 0.83 for FC and 0.86 for IF. These results imply that carabrone exerts its antifungal activity against Ggt by interfering with mitochondrial function.
[Mh] Termos MeSH primário: Antifúngicos/farmacologia
Ascomicetos/efeitos dos fármacos
Fungicidas Industriais/farmacologia
Mitocôndrias/efeitos dos fármacos
[Mh] Termos MeSH secundário: Animais
Antifúngicos/química
Ascomicetos/metabolismo
Feminino
Imunofluorescência/métodos
Fungicidas Industriais/síntese química
Fungicidas Industriais/química
Haptenos/química
Hifas/efeitos dos fármacos
Hifas/ultraestrutura
Cinética
Camundongos Endogâmicos BALB C
Mitocôndrias/ultraestrutura
Imagem Molecular/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antifungal Agents); 0 (Fungicides, Industrial); 0 (Haptens)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180215
[Lr] Data última revisão:
180215
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171214
[St] Status:MEDLINE
[do] DOI:10.1021/acs.jafc.7b03913


  10 / 7526 MEDLINE  
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[PMID]:29273507
[Au] Autor:Furuya T; Takehara I; Shimura A; Kishimoto H; Yasujima T; Ohta K; Shirasaka Y; Yuasa H; Inoue K
[Ad] Endereço:Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
[Ti] Título:Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.
[So] Source:Biochem Biophys Res Commun;495(3):2152-2157, 2018 01 15.
[Is] ISSN:1090-2104
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K ) of 0.23 µM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent K of 0.36 µM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.
[Mh] Termos MeSH primário: Benzotiazóis/metabolismo
Aumento da Imagem/métodos
Luciferases/metabolismo
Medições Luminescentes/métodos
Proteína 1 Transportadora de Ânions Orgânicos/metabolismo
[Mh] Termos MeSH secundário: Genes Reporter/genética
Células HEK293
Seres Humanos
Luciferases/genética
Imagem Molecular/métodos
Reprodutibilidade dos Testes
Sensibilidade e Especificidade
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Benzothiazoles); 0 (D-luciferin); 0 (Organic Anion Transport Protein 1); EC 1.13.12.- (Luciferases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180213
[Lr] Data última revisão:
180213
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171224
[St] Status:MEDLINE



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