RESUMO
Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 µM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only â¼1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at â¼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (â¼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98±0.07×10(-7)c m(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was â¼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.
Assuntos
Laranja de Acridina/química , Exocitose , Corantes Fluorescentes/química , Mucinas/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Cinética , Microscopia de Fluorescência , Mucinas/química , Imagem Óptica , Sus scrofaRESUMO
A cationic azadioxatriangulenium (ADOTA) dye was entrapped in silica thin films obtained by the sol-gel process and in poly (vinyl) alcohol (PVA) thin films. Azadioxatriangulenium is a red emitting fluorophore with a long fluorescence lifetime of ~20 ns. The fluorescent properties of azadioxatriangulenium in silica thin films and PVA films were studied by means of steady-state and time resolved fluorescence techniques. We have found that the azadioxatriangulenium entrapped in silica thin film has a wider fluorescence lifetime distribution (Lorentzian distribution), lower fluorescence efficiencies, shorter lifetimes compared to Azadioxatriangulenium in a PVA film. The local environment of azadioxatriangulenium molecules in the silica thin film is rich with water and ethanol, which creates the possibility of forming excited state aggregates due to high concentration of dye within a small confined area. In contrast to the PVA matrices, the porous silica films allow restricted rotations of Azadioxatriangulenium molecules, which result in faster and complex fluorescence anisotropy decays suggesting energy migration among dye molecules.
RESUMO
Sigma-1 receptors (σ-1rs) exert neuroprotective effects on retinal ganglion cells (RGCs) both in vivo and in vitro. This receptor has unique properties through its actions on several voltage-gated and ligand-gated channels. The purpose of this study was to investigate the role that σ-1rs play in regulating cell calcium dynamics through activated L-type Voltage Gated Calcium Channels (L-type VGCCs) in purified RGCs. RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using a Thy1.1 antibody. Calcium imaging was used to measure changes in intracellular calcium after depolarizing the cells with potassium chloride (KCl) in the presence or absence of two σ-1r agonists [(+)-SKF10047 and (+)-Pentazocine], one σ-1r antagonist (BD1047), and one L-type VGCC antagonist (Verapamil). Finally, co-localization studies were completed to assess the proximity of σ-1r with L-type VGCCs in purified RGCs. VGCCs were activated using KCl (20 mM). Pre-treatment with a known L-type VGCC blocker demonstrated a 57% decrease of calcium ion influx through activated VGCCs. Calcium imaging results also demonstrated that σ-1r agonists, (+)-N-allylnormetazocine hydrochloride [(+)-SKF10047] and (+)-Pentazocine, inhibited calcium ion influx through activated VGCCs. Antagonist treatment using BD1047 demonstrated a potentiation of calcium ion influx through activated VGCCs and abolished all inhibitory effects of the σ-1r agonists on VGCCs, implying that these ligands were acting through the σ-1r. An L-type VGCC blocker (Verapamil) also inhibited KCl activated VGCCs and when combined with the σ-1r agonists there was not a further decline in calcium entry suggesting similar mechanisms. Lastly, co-localization studies demonstrated that σ-1rs and L-type VGCCs are co-localized in purified RGCs. Taken together, these results indicated that σ-1r agonists can inhibit KCl induced calcium ion influx through activated L-type VGCCs in purified RGCs. This is the first report of attenuation of L-type VGCC signaling through the activation of σ-1rs in purified RGCs. The ability of σ-1rs to co-localize with L-type VGCCs in purified RGCs implied that these two proteins are in close proximity to each other and that such interactions regulate L-type VGCCs.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Receptores sigma/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Western Blotting , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Etilenodiaminas/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Fura-2/análogos & derivados , Fura-2/metabolismo , Microscopia de Fluorescência , Pentazocina/farmacologia , Fenazocina/análogos & derivados , Fenazocina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores sigma/agonistas , Receptores sigma/antagonistas & inibidores , Células Ganglionares da Retina/metabolismo , Verapamil/farmacologia , Receptor Sigma-1RESUMO
Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca(2+) entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca(2+) entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca(2+) entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H(2)O(2) itself evoked Ca(2+) influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H(2)O(2) also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H(2)O(2) stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H(2)O(2) triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.
Assuntos
Células Musculares/metabolismo , Músculo Liso Vascular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Aorta Torácica , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/farmacologia , Canal de Cátion TRPC6 , Rigidez Vascular/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.
RESUMO
In the present work, we have synthesized water soluble Ag nanoclusters using PMAA as a template with different Ag+: COO-ratios, to optimize it for highest brightness using less UV exposure time. Fluorescence polarization was 0.30 for and was found to vary with excitation and emission wavelength with few hundred picoseconds average fluorescence lifetime. Fluorescence Correlation Spectroscopy data depicts slower diffusion at red excitation compared to blue excitation in confocal volume than conventionally synthesized colloids proving presence of multiple sizes. The optical properties of the particles are dependent upon the excitation wavelength used and the emission wavelength collected.
RESUMO
Studies on mice showed that chitosan as an adjuvant for H5 inactivated influenza vaccines administered intramuscularly enhances significantly antibody titers and protective efficiency not only against homologous influenza viruses, but also against drift variants. Chitosan adjuvanted vaccines induced high antibody titers after a single immunization and with a low dose of antigen. High antibody titers remained for at least 6 months. Chitosan adjuvanted vaccine stored at 4 degrees C preserves its adjuvant properties for at least 8 months. Chitosan stimulates proliferative and cytotoxic activity of splenic mononuclear leukocytes in mice and promotes an increase in the numbers of CD3, CD3/NK, I-AK (MHC II), and H-2Db (MHC I) cells. After intramuscular immunization, chitosan did not induce IgE antibodies and antibodies against chitosan itself. Chitosan is a promising adjuvant candidate for inactivated influenza vaccines administered parenterally.
Assuntos
Adjuvantes Imunológicos/farmacologia , Quitosana/farmacologia , Vacinas contra Influenza/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Proliferação de Células , Quitosana/administração & dosagem , Citocinas/metabolismo , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Imunoglobulina E/sangue , Vacinas contra Influenza/administração & dosagem , Injeções Intramusculares , Leucócitos Mononucleares/imunologia , Camundongos , Coelhos , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Temperatura , Vacinas de Produtos Inativados/imunologiaRESUMO
We report a multi-fold enhancement of the fluorescence of methyl-azadioxatriangulenium chloride (Me-ADOTA*Cl) in PVA deposited on a 50 nm thick gold mirror carrying an evaporation induced self-assembly of colloidal silver nanoparticles (Ag-SACs). The average measured increase in fluorescence emission of about 50-fold is accompanied by hot spots with a local enhancement in brigthness close to 200. The long lifetime of the dye allows for the first direct determination of the correlation between the enhancement of emission intensity and the decrease in fluorescence lifetime. The Ag-SACs surface preparation and observed enhancements are highly reproducible. We believe that these robust plasmonic surfaces will find use in sensing platforms for ultrasensitive detection.
RESUMO
Recently it has become possible to study single protein molecules in a cell. However, such experiments are plagued by rapid photobleaching. We recently showed that the interaction of fluorophores with localized surface plasmon polaritons (LSPs) induced in the metallic nanoparticles led to a substantial reduction of photobleaching. We now investigate whether the photobleaching could be further reduced when the excited fluorophore interacts with the LSP excited in the metallic nanoparticles resident on mirrored surface. As an example we use myofibrils, subcellular structures within skeletal muscle. We compare nanoparticle-enhanced fluorescence of myofibrils in the presence and in the absence of a mirrored surface. The proximity of the mirrored surface led to enhancement of fluorescence and to a decrease in fluorescent lifetime, much greater than that observed in the presence of nanoparticles alone. We think that the effect is caused by the near-field interactions between fluorophores and LSP, and between fluorophores and propagating surface plasmons (PSPs) produced in the metallic surface by the nanoparticles. Photobleaching is decreased because fluorescence enhancement enables illumination with a weaker laser beam and because the decrease in fluorescence lifetime minimizes the probability of oxygen attack during the time a molecule is in the exited state.
Assuntos
Microscopia de Fluorescência/métodos , Proteínas Musculares/análise , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Nanopartículas , Prata , Ressonância de Plasmônio de Superfície/métodos , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência/métodos , Aumento da Imagem/métodos , Fotodegradação , CoelhosRESUMO
Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.
Assuntos
Actinas/efeitos da radiação , Actinas/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Técnicas de Sonda Molecular , Miofibrilas/efeitos da radiação , Miofibrilas/ultraestrutura , Actinas/metabolismo , Animais , Artefatos , Humanos , Aumento da Imagem/métodos , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos da radiação , Miofibrilas/metabolismo , Fotodegradação/efeitos da radiação , UltrassonografiaRESUMO
Gold nanoparticles covalently attached to the indium tin oxide coated glass slide drastically quench fluorescence of a surface immunoassay (approximately 5-fold). Silver electrochemically deposited over the gold particles leads to fluorescence amplification: signal increases approximately 7-8 times if compared to the signal on gold particles not covered with silver. This phenomenon allows enhancing of the surface immunoassays utilizing both types of nanoparticles.
RESUMO
Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient). This work introduces such a method, which analyzes fluctuations of anisotropy of a few actin molecules in muscle. Fluorescence anisotropy, indicating orientation of an actin protomer, is collected from a volume of a few attoliters (10(-18) L) by confocal total internal reflection (CTIR) microscopy. In this method, the detection volume is made shallow by TIR illumination, and narrow by confocal aperture inserted in the conjugate image plane. The signal is contributed by approximately 12 labeled actin molecules. Shortening of a myofibril during contraction is prevented by light cross-linking with 1-ethyl-3-[3-dimethylamino)-propyl]-carbodiimide. The root mean-squared anisotropy fluctuations are greater in isometrically contracting than in rigor myofibrils. The results support the view that during isometric contraction, cross-bridges undergo a mechanical cycle.
Assuntos
Actinas/fisiologia , Actinas/ultraestrutura , Contração Isométrica/fisiologia , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Proteínas Motores Moleculares/fisiologia , Proteínas Motores Moleculares/ultraestrutura , Conformação Proteica , RotaçãoRESUMO
Tumor-derived exosomes are emerging mediators of tumorigenesis and tissue-specific metastasis. Proteomic profiling has identified Annexin II as one of the most highly expressed proteins in exosomes; however, studies focused on the biological role of exosomal Annexin II (exo-Anx II) are still lacking. In this study, mechanistic insight was sought regarding exo-Anx II and its function in angiogenesis and breast cancer metastasis. Multiple in vitro and in vivo techniques were used to study the role of exo-Anx II in angiogenesis. Using atomic force microscopy and Western blotting, exo-Anx II expression was characterized in normal and breast cancer cells. In addition, organ-specific metastatic breast cancer cells and animal models were used to define the role exo-Anx II in breast cancer metastasis. Results revealed that exo-Anx II expression is significantly higher in malignant cells than normal and premetastatic breast cancer cells. In vitro and in vivo studies demonstrated that exo-Anx II promotes tPA-dependent angiogenesis. Furthermore, in vivo analysis indicated that metastatic exosomes create a favorable microenvironment for metastasis, and exo-Anx II plays an important role in this process, as priming with Anx II-depleted exosomes reduces brain (â¼4-fold) and lung (â¼2-fold) metastasis. Upon delineating the mechanism, it was discovered that exo-Anx II causes macrophage-mediated activation of the p38MAPK, NF-κB, and STAT3 pathways and increased secretion of IL6 and TNFα. These data demonstrate an important role for exo-Anx II in breast cancer pathogenesis. IMPLICATIONS: Exosome-associated Annexin II plays an important role in angiogenesis and breast cancer metastasis, which can be exploited as a potential biomarker as well as a therapeutic target for diagnosis and treatment of metastatic breast cancer. Mol Cancer Res; 15(1); 93-105. ©2016 AACR.
Assuntos
Anexina A2/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Exossomos/metabolismo , Neovascularização Patológica/metabolismo , Animais , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , NF-kappa B/metabolismo , Neovascularização Patológica/patologia , Fótons , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tecidual/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter). By restraining labeling to 1 fluorophore per 100 myosin molecules, we were able to follow the kinetics of approximately 400 cross-bridges. To reduce this number further, we used two-photon (2P) microscopy. The focal plane in which the laser power density was high enough to produce 2P absorption was thinner than in confocal microscopy. Using 2P microscopy, we were able to observe approximately 200 cross-bridges during contraction. The novel method of confocal total internal reflection (CTIR) provides a method to reduce the observational volume even further, to approximately 1 attoliter (10(-18) liter), and to measure fluorescence with a high signal-to-noise (S/N) ratio. In this method, the observational volume is made shallow by illuminating the sample with an evanescent field produced by total internal reflection (TIR) of the incident laser beam. To guarantee the small lateral dimensions of the observational volume, a confocal aperture is inserted in the conjugate-image plane of the objective. With a 3.5-mum confocal aperture, we achieved a volume of 1.5 attoliter. Association-dissociation of the myosin head was probed with rhodamine attached at cys707 of the heavy chain of myosin. Signal was contributed by one to five fluorescent myosin molecules. Fluorescence decayed in a series of discrete steps, corresponding to bleaching of individual molecules of rhodamine. The S/N ratio was sufficiently large to make statistically significant comparisons from rigor and contracting myofibrils.
Assuntos
Proteínas Motores Moleculares/fisiologia , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Miosinas/fisiologia , Corantes Fluorescentes/química , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Motores Moleculares/ultraestrutura , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Miosinas/ultraestruturaRESUMO
In this report we describe a preparation of silver wires (SWs) on gold mirrors and its application to surface enhanced fluorescence (SEF) using a new methodology. Silica protected gold mirrors were drop-coated with a solution of silver triangular nanoprisms. The triangular nanoprisms were slowly air-dried to get silver wires that self-assembled on the gold mirrors. Fluorescence enhancement was studied using methyl azadioxatriangulenium chloride (Me-ADOTA · Cl) dye in PVA spin-coated on a clean glass coverslip. New Plasmonic Platforms (PPs) were assembled by placing a mirror with SWs in contact with a glass coverslip spin-coated with a uniform Me-ADOTA · Cl film. It was shown that surface enhanced fluorescence is a real phenomenon, not just an enhancement of the fluorescence signal due to an accumulation of the fluorophore on rough nanostructure surfaces. The average fluorescence enhancement was found to be about 15-fold. The lifetime of Me-ADOTA · Cl dye was significantly reduced (â¼ 4 times) in the presence of SWs. Moreover, fluorescence enhancement and lifetime did not show any dependence on the excitation light polarization.
Assuntos
Corantes Fluorescentes/química , Ouro/química , Microscopia de Fluorescência/métodos , Nanofios/química , Prata/química , Ressonância de Plasmônio de Superfície/métodosRESUMO
Fluorescent nanodiamonds (FNDs) are one of the new and very promising biocompatible nanomaterials that can be used both as a fluorescence imaging agent and a highly versatile platform for controlled functionalization to target and deliver a wide spectrum of therapeutic agents. Among the remarkable fluorescence properties are excellent photostability, emission between 600-700nm, quantum yield of 1 and moderately long fluorescence lifetimes. However the low absorption cross section of fluorescent (N-V)(-) centers limits FNDs' brightness. In this work we show that an approach based on the Forster resonance energy transfer (FRET) may significantly enhance the fluorescence signal observed from a single ND. We demonstrate that organic dyes (fluorophores) attached to the FND surface can efficiently transfer the excitation energy to (N-V)(-) centers. Multiple dyes positioned in close proximity to the ND facile surface may serve as harvesting antennas transferring excitation energy to the fluorescent centers. We propose that, with the help of some of the functional groups present on the FND surface, we can either directly link flurophores or use scalable dendrimer chemistry to position many organic dyes at a calibrated distance. Also, the remaining multiple functional groups will be still available for particle targeting and drug delivery. This opens a new way for designing a new type of theranostics particles of ultrahigh brightness, high photostability, specific targeting, and high capacity for drug delivery.
Assuntos
Diamante/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes , Nanoestruturas/química , Fluoresceína-5-Isotiocianato , Fluorescência , Microscopia Confocal , Soroalbumina Bovina/química , Espectrofotometria UltravioletaRESUMO
CS1 (CD319, CRACC, SLAMF7, novel Ly9) activates NK cell-mediated cytotoxicity and proliferation of B lymphocytes during immune responses. The expression of CS1 is up regulated on B cells in multiple myeloma and systemic lupus erythematosus. In this study we describe the transcriptional regulation of mouse CS1 (mCS1) gene. We show that mCS1 gene transcription is regulated by YY1 (Ying Yang 1) and a unique (AG)n=36 DNA repeat element. YY1 is known to play a significant role in B cell development by regulating the pro B cell to pre B cell transition. The consensus DNA binding site for YY1 was detected using TRANSFAQ on the mCS1 promoter region. Mutations in the YY1 site led to a significant increase in mCS1 promoter activity indicating that YY1 represses mCS1 transcription. YY1 binds to the mCS1 promoter at the expected site in vivo and in vitro as tested by chromatin immunoprecipitation assays and super-shift EMSA assays respectively. Unique (CT)n=24 and (AG)n=36 DNA repeat elements are present on mCS1 promoter that are sensitive to S1 nuclease and engage in DNA triplex structure as confirmed by AFM (atomic force microscopy) imaging. Interestingly, the (AG)n=36 repeat element enhances mCS1 promoter activity.
Assuntos
Receptores Imunológicos/genética , Sequências Repetitivas de Ácido Nucleico , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Células Precursoras de Linfócitos B/metabolismo , Regiões Promotoras Genéticas , Receptores Imunológicos/metabolismo , Transcrição Gênica , Ativação Transcricional , Regulação para CimaRESUMO
Despite major advances in pediatric cancer research, there has been only modest progress in the survival of children with high risk neuroblastoma (NB) (HRNB). The long term survival rates of HRNB in the United States are still only 30-50%. Due to resistance that often develops during therapy, development of new effective strategies is essential to improve the survival and overcome the tendency of HRNB patients to relapse subsequent to initial treatment. Current chemotherapy regimens also have a serious limitation due to off target toxicity. In the present work, we evaluated the potential application of reconstituted high density lipoprotein (rHDL) containing fenretinide (FR) nanoparticles as a novel approach to current NB therapeutics. The characterization and stability studies of rHDL-FR nanoparticles showed small size (<40 nm) and high encapsulation efficiency. The cytotoxicity studies of free FR vs. rHDL/FR toward the NB cell lines SK-N-SH and SMS-KCNR showed 2.8- and 2-fold lower IC50 values for the rHDL encapsulated FR vs. free FR. More importantly, the IC50 value for retinal pigment epithelial cells (ARPE-19), a recipient of off target toxicity during FR therapy, was over 40 times higher for the rHDL/FR as compared to that of free FR. The overall improvement in in vitro selective therapeutic efficiency was thus about 100-fold upon encapsulation of the drug into the rHDL nanoparticles. These studies support the potential value of this novel drug delivery platform for treating pediatric cancers in general, and NB in particular.
RESUMO
The surface-confined assay format is one of the most convenient detection formats used in many immunoassays. Fluorescence emission from monolayers of dyes requires a strong excitation and good detection system. Such samples are susceptible to artifacts due to background fluorescence from substrates. We demonstrate that using silver nanostructures deposited on the slide substrate can significantly enhance measured fluorescence, reduce unwanted background and increase photostability of the used probes. Using thin layers of polymer doped with fluorescein, we tested two nanostructures--silver island films (SIFs) deposited on glass slides and self-assembled colloidal structures (SACS) deposited on thin silver film. The SACS surfaces show extraordinary fluorescence enhancements: over 100-folds in hot spots. We applied these surfaces for enhanced Alexa488 model immunoassay.
Assuntos
Imunoensaio/métodos , Metais/química , Imunofluorescência/métodos , Nanopartículas Metálicas/química , Microscopia de Força Atômica/métodos , Prata/química , Espectrometria de Fluorescência/métodosRESUMO
Total internal reflection fluorescence microscopy (TIRFM) has been utilized to explore the effect of cationic protein lysozyme (Lz) on the morphology of solid-supported lipid bilayers (SLBs) comprised of zwitterionic lipid phosphatidylcholine (PC) and its mixture with anionic lipid cardiolipin (CL). Kinetic TIRFM imaging of different systems revealed subtle interplay between lipid lateral segregation accompanied by exchange of neutral and acidic lipids in the protein-lipid interaction zone, and the formation of lipid multilayer stacks. The switch between these states was shown to be controlled by CL content. In weakly charged SLBs containing 5 mol% CL, assembling of CL molecules into planar domains upon Lz adsorption has been observed while at higher content of anionic lipid (25 mol%) in-plane domains tend to transform into multilayer stacks, thereby ensuring the most thermodynamically-favorable membrane conformation.