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1.
Nat Commun ; 11(1): 4285, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855390

RESUMO

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citocininas/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brefeldina A/farmacologia , Citocininas/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Meristema/citologia , Meristema/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
PLoS One ; 15(7): e0236171, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702047

RESUMO

Cell-substrate adhesion of the social amoeba Dictyostelium discoideum, a model organism often used for the study of chemotaxis, is non-specific and does not involve focal adhesion complexes. Therefore, micropatterned substrates where adherent Dictyostelium cells are constrained to designated microscopic regions are difficult to make. Here we present a micropatterning technique for Dictyostelium cells that relies on coating the substrate with an ∼1µm thick layer of polyethylene glycol (PEG) gel. We show that, when plated on a substrate with narrow parallel stripes of PEG-gel and glass, Dictyostelium cells nearly exclusive adhere to and migrate along the glass stripes, thus providing a model system to study one-dimensional migration of amoeboid cells. Surprisingly, we find substantial differences in the adhesion to PEG-gel and glass stripes between vegetative and developed cells and between two different axenic laboratory strains of Dictyostelium, AX2 and AX4. Even more surprisingly, we find that the distribution of Dictyostelium cells between PEG-gel and glass stripes is significantly affected by the expression of several fluorescent protein markers of the cytoskeleton. We carry out atomic force microscopy based single cell force spectroscopy measurements that confirm that the force of adhesion to PEG-gel substrate can be significantly different between vegetative and developed cells, AX2 and AX4 cells, and cells with and without fluorescent markers. Thus, the choice of parental background, the degree of development, and the expression of fluorescent protein markers can all have a profound effect on cell-substrate adhesion and should be considered when comparing migration of cells and when designing micropatterned substrates.


Assuntos
Movimento Celular , Dictyostelium/citologia , Corantes Fluorescentes/metabolismo , Microtecnologia/métodos , Polietilenoglicóis/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dictyostelium/efeitos dos fármacos , Géis/farmacologia , Análise Espectral
3.
J Vis Exp ; (160)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32568221

RESUMO

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Compostos de Boro/metabolismo , Sobrevivência Celular , Cor , Ácidos Graxos/metabolismo , Corantes Fluorescentes/metabolismo , Gotículas Lipídicas/metabolismo , Lisossomos/metabolismo , Leveduras/citologia
4.
Proc Natl Acad Sci U S A ; 117(26): 14694-14702, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554491

RESUMO

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.


Assuntos
Encéfalo/enzimologia , DNA/química , Corantes Fluorescentes/química , Óxido Nítrico Sintase Tipo II , Animais , Encéfalo/metabolismo , Química Encefálica , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/química , Microglia/enzimologia , Microglia/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fagossomos/química , Fagossomos/metabolismo , Peixe-Zebra
5.
Nat Commun ; 11(1): 3216, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32587248

RESUMO

Chemical tools to monitor drug-target engagement of endogenously expressed protein kinases are highly desirable for preclinical target validation in drug discovery. Here, we describe a chemical genetics strategy to selectively study target engagement of endogenous kinases. By substituting a serine residue into cysteine at the DFG-1 position in the ATP-binding pocket, we sensitize the non-receptor tyrosine kinase FES towards covalent labeling by a complementary fluorescent chemical probe. This mutation is introduced in the endogenous FES gene of HL-60 cells using CRISPR/Cas9 gene editing. Leveraging the temporal and acute control offered by our strategy, we show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis via SYK kinase activation. This chemical genetics strategy holds promise as a target validation method for kinases.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes , Proteínas Proto-Oncogênicas c-fes , Transportadores de Cassetes de Ligação de ATP/química , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Edição de Genes , Humanos , Macrófagos/metabolismo , Mutação , Neutrófilos , Fagocitose , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fes/química , Proteínas Proto-Oncogênicas c-fes/genética , Proteínas Proto-Oncogênicas c-fes/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo
6.
J Vis Exp ; (160)2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32597871

RESUMO

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.


Assuntos
Envelhecimento/fisiologia , Bioensaio/métodos , Drosophila melanogaster/citologia , Hemócitos/citologia , Fagócitos/citologia , Fagocitose , Animais , Dissecação , Feminino , Corantes Fluorescentes/metabolismo , Hemócitos/metabolismo , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Injeções , Fagócitos/metabolismo , Coloração e Rotulagem , Fixação de Tecidos
7.
J Med Chem ; 63(10): 5297-5311, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32420741

RESUMO

Comprehensively characterized fluorescent probes for the histamine H3 receptor (H3R) and especially for the H4R orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementary tools to radioligands. In view of fluorescent probes for BRET-based binding studies and for localizing the H4R in live cells, we synthesized and biologically characterized Py-5-labeled histamine derivatives. The most notable compound was UR-DEBa242 (26, 1-[4-(1H-Imidazol-4-yl)butyl]-4-{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}-2,6-dimethylpyridinium hydrotrifluoroacetate trifluoroacetate), acting as a partial agonist at the hH3R [pEC50 (reporter gene) 8.77] and as an inverse agonist/antagonist at the h/mH4Rs [pIC50 (reporter gene) 8.76/7.08; pIC50/pKb (ß-arrestin2) 7.81/7.30]. In confocal microscopy, 26 proved suitable for hH4R localization and trafficking studies in live cells. BRET-based binding at the NLuc-hH3,4Rs/mH4R [pKd 8.78/7.75/7.18, comparable to binding constants from radioligand binding/flow cytometry; fast association/dissociation (∼2 min)] revealed 26 as a useful molecular tool to determine hH3,4Rs/mH4R binding affinities of ligands binding to these receptors.


Assuntos
Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Receptores Histamínicos H3/análise , Receptores Histamínicos H3/metabolismo , Receptores Histamínicos H4/análise , Receptores Histamínicos H4/metabolismo , Animais , Células HEK293 , Humanos , Células Sf9
8.
BMC Infect Dis ; 20(1): 303, 2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321447

RESUMO

BACKGROUND: This study aimed to establish and evaluate a simultaneous amplification and testing method for detection of extra-pulmonary tuberculosis (EPTB). METHODS: From January 2016 and December 2017 the pus or surgical excision from the lesions of inpatients admitted from Chongqing Public Health Treatment Center were collected. According to the clinical diagnosis, the samples were divided into two groups including EPTB (Group A) and other diseases excluded from tuberculosis diseases (Group B). Simultaneous detection of Mycobacterium tuberculosis (MTB) used Roche culture method, liquid culture method and simultaneous amplification and testing (SAT) method. The sensitivity and specificity of the SAT method were compared with culture methods and clinical diagnosis of EPTB. RESULTS: For 433 EPTB specimens and 49 non-TB specimens, the simultaneous amplification and testing tuberculosis (SAT-TB) results correlated with 80.5% (388/482 specimens) of the culture assay results. The sensitivity, specificity, and positive and negative predictive values of the SAT-TB test for the diagnosis of EPTB were 83.6, 79.4, 59.4, and 93.0%, respectively, compared to culture methods. Compared with the clinical diagnosis of patients, the sensitivity and specificity of the SAT-TB test were 41.6 and 100%, respectively, the cultures test were 29.3 and 98.0%. CONCLUSIONS: SAT test is a simple and rapid test with high specificity which may enhance the detection of EPTB. SAT-TB is a higher clinical diagnosis value for EPTB in clinical microbiology laboratories.


Assuntos
Corantes Fluorescentes/análise , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose/diagnóstico , Reações Falso-Negativas , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Técnicas Microbiológicas/métodos , Testes Imediatos , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Tuberculose/microbiologia
9.
Org Biomol Chem ; 18(15): 2938-2948, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32242600

RESUMO

Two myo-inositol derivatives having an Nα,Nε-diacetyl-l-lysine (Ac2Lys) moiety linked to the inositol 1-O-position through a self-cleavable linker and a metabolically stable 2-azidoethyl group linked to the inositol 3-O- and 4-O-positions, respectively, were designed and synthesized. The Ac2Lys moiety blocking the inositol 1-O-position required for GPI biosynthesis was expected to be removable by a combination of two enzymes, histone deacetylase (HDAC) and cathepsin L (CTSL), abundantly expressed in cancer cells, but not in normal cells, to transform these inositol derivatives into biosynthetically useful products with a free 1-O-position. As a result, it was found that these inositol derivatives could be incorporated into the glycosylphosphatidylinositol (GPI) biosynthetic pathway by cancer cells, but not by normal cells, to express azide-labeled GPIs and GPI-anchored proteins on cell surfaces. Consequently, this study has established a novel strategy and new molecular tools for selective metabolic labeling of cancer cells, which should be useful for various biological studies and applications.


Assuntos
Corantes Fluorescentes/química , Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Inositol/química , Lisina/química , Engenharia Metabólica , Imagem Óptica , Células Cultivadas , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Proteínas Ligadas por GPI/química , Glicosilfosfatidilinositóis/química , Células HEK293 , Humanos , Inositol/síntese química , Inositol/metabolismo , Lisina/síntese química , Lisina/metabolismo , Microscopia de Fluorescência
10.
J Med Chem ; 63(8): 3996-4004, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32227886

RESUMO

Constitutive proteolytic activity of MALT1 is associated with highly aggressive B-cell lymphomas. Chemical tools that detect active MALT1 have been reported, but suffer from poor cell permeability and/or cross-reactivity with the cysteine protease cathepsin B. Here, we report that the non-natural amino acid pipecolinic acid in the P2 position of substrates and chemical probes leads to improved selectivity toward MALT1 and results in cell-permeable fluorescent probes.


Assuntos
Aminoácidos/síntese química , Aminoácidos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Aminoácidos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/fisiologia , Desenho de Fármacos , Corantes Fluorescentes/farmacologia , Humanos , Células Jurkat , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
11.
J Med Chem ; 63(8): 4133-4154, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32233403

RESUMO

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.


Assuntos
Corantes Fluorescentes/metabolismo , Antagonistas Muscarínicos/metabolismo , Ftalimidas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M2/metabolismo , Animais , Células CHO , Colinérgicos/química , Colinérgicos/metabolismo , Colinérgicos/farmacologia , Cricetulus , Corantes Fluorescentes/química , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/farmacologia , Ftalimidas/química , Ftalimidas/farmacologia , Estrutura Secundária de Proteína , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia
12.
AAPS PharmSciTech ; 21(4): 122, 2020 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-32337617

RESUMO

This study aimed to investigate the effect of pH-mediated surface properties of bovine serum albumin (BSA) on protein aggregation and the changes of protein structure and colloidal stability at different solution pH levels. The hydrophobicity of BSA surface was characterized by endogenous fluorescence spectroscopy, fluorescence quenching of acrylamide, and fluorescence probe. The results showed that the hydrophobicity of BSA surface was similar at pH 5, 6, 7.4, followed by pH 4, 8, 9, 10, and finally by pH 3 and 11 with strong acidity and alkalinity. The positive charge on the BSA surface was increased gradually with the decrease of solution pH, while the negative charge on protein surface was increased gradually with the increase of solution pH. The degree of protein aggregation was examined by turbidimetry, flow cytometry, and SDS-PAGE. The results showed that the oscillating aggregation of BSA did not change with the solution pH, but was partially dependent on the relative contribution of electrostatic and hydrophobic interactions between the protein molecules. In addition, the secondary structure, conformational stability, unfolding degree, and colloidal stability of proteins were investigated by circular dichroism, fluorescence spectroscopy, protein pulse hydrolysis, and dynamic light scattering, respectively. The results suggested that the solution pH could change the structure and stability of the protein at different levels. Solution pH has distinct effects on the structural stability of protein at different levels. The change of protein surface properties mediated by solution pH is related to protein aggregation.


Assuntos
Agregados Proteicos , Soroalbumina Bovina/química , Água/química , Animais , Bovinos , Dicroísmo Circular/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência/métodos , Eletricidade Estática , Propriedades de Superfície , Água/metabolismo
13.
J Vis Exp ; (157)2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32225141

RESUMO

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.


Assuntos
Sistemas de Secreção Bacterianos , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Legionella pneumophila/ultraestrutura , Viabilidade Microbiana , Alelos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Citosol/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Subunidades Proteicas/metabolismo , Recombinação Genética/genética
14.
J Med Chem ; 63(7): 3563-3576, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32207938

RESUMO

In an effort to seek novel agents targeting prostate-specific membrane antigen (PSMA), 16 ligands (L1-L16) with structural modifications in S1' binding pocket were synthesized and evaluated for PSMA inhibition. (S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic acids proved to be potent PSMA ligands with Ki values ranging from 0.08 nM to 8.98 nM, which are in the range of or are higher in potency compared to previously published urea-based ligands. Computational docking was performed to study the binding mode of the two most potent ligands discovered. FITC-conjugated L14 could selectively stain PSMA+ LNCaP cells over PSMA- PC3 cells. IRDye800CW conjugated L16 can effectively image tumors in a murine xenograft model of prostate cancer.


Assuntos
Corantes Fluorescentes/farmacologia , Neoplasias da Próstata/diagnóstico por imagem , Ureia/análogos & derivados , Ureia/farmacologia , Animais , Antígenos de Superfície/metabolismo , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Imagem Óptica/métodos , Estudo de Prova de Conceito , Ligação Proteica , Ureia/metabolismo
15.
Chemistry ; 26(28): 6279-6284, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32154932

RESUMO

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.


Assuntos
Trifosfato de Adenosina/química , Corantes Fluorescentes/química , Enzimas Ativadoras de Ubiquitina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Enzimas Ativadoras de Ubiquitina/química
16.
Nat Commun ; 11(1): 1250, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144257

RESUMO

Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).


Assuntos
Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Miosite/diagnóstico por imagem , Infecção da Ferida Cirúrgica/diagnóstico por imagem , Trissacarídeos/administração & dosagem , Animais , Carbocianinas/administração & dosagem , Carbocianinas/química , Modelos Animais de Doenças , Estabilidade de Medicamentos , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Injeções Intravenosas , Medições Luminescentes/métodos , Camundongos , Microscopia de Fluorescência/métodos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Miosite/microbiologia , Técnicas Fotoacústicas/métodos , Ratos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Infecção da Ferida Cirúrgica/microbiologia , Trissacarídeos/química , Trissacarídeos/metabolismo
18.
Chem Commun (Camb) ; 56(17): 2598-2601, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32016205

RESUMO

A novel pyrene-based naked-eye colorimetric and fluorescent turn-on probe (S-ClO) was developed. Besides, imaging in living RAW264.7 cells, an arterial vessel inflammation nude mouse model and human serum using S-ClO indicates that S-ClO could serve as a potential tool for early diagnosing and monitoring inflammatory diseases.


Assuntos
Aterosclerose/metabolismo , Corantes Fluorescentes/metabolismo , Ácido Hipocloroso/metabolismo , Animais , Camundongos , Camundongos Nus , Microscopia de Fluorescência/métodos , Células RAW 264.7
19.
Chem Commun (Camb) ; 56(18): 2731-2734, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32022000

RESUMO

We herein develop two ß-galactosidase (ß-Gal) activatable NIR fluorescent probes for visualizing ovarian cancers. Particularly, probe BOD-M-ßGal produced NIR-II emission light at 900-1300 nm upon ß-Gal activation. By using our activatable and target specific NIR-II probe for deep-tissue imaging of ß-Gal overexpressed ovarian cancer cells, rapid and accurate imaging of ovarian tumors in nude mice was achieved.


Assuntos
Corantes Fluorescentes/química , Imagem Óptica , Neoplasias Ovarianas/diagnóstico por imagem , beta-Galactosidase/química , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Corantes Fluorescentes/metabolismo , Células Endoteliais da Veia Umbilical Humana/química , Humanos , Raios Infravermelhos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/metabolismo , beta-Galactosidase/metabolismo
20.
Nat Commun ; 11(1): 793, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034152

RESUMO

Fluorescence-based technologies have revolutionized in vivo monitoring of biomolecules. However, significant technical hurdles in both probe chemistry and complex cellular environments have limited the accuracy of quantifying these biomolecules. Herein, we report a generalizable engineering strategy for dual-emission anti-Kasha-active fluorophores, which combine an integrated fluorescein with chromene (IFC) building block with donor-π-acceptor structural modification. These fluorophores exhibit an invariant near-infrared Kasha emission from the S1 state, while their anti-Kasha emission from the S2 state at around 520 nm can be finely regulated via a spirolactone open/closed switch. We introduce bio-recognition moieties to IFC structures, and demonstrate ratiometric quantification of cysteine and glutathione in living cells and animals, using the ratio (S2/S1) with the S1 emission as a reliable internal reference signal. This de novo strategy of tuning anti-Kasha-active properties expands the in vivo ratiometric quantification toolbox for highly accurate analysis in both basic life science research and clinical applications.


Assuntos
Bioquímica/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Células A549 , Animais , Benzopiranos/química , Cisteína/análise , Feminino , Fluoresceína/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Glutationa/análise , Células Hep G2 , Humanos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Piranos/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Espironolactona/química
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