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Find video protocols related to scientific articles indexed in Pubmed.
A Biphenyl Type Two-Photon Fluorescence Probe for Monitoring the Mitochondrial Membrane Potential.
Cell Struct. Funct.
PUBLISHED: 10-17-2014
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Here we describe the design and synthesis of a bifunctional two-photon fluorescence probe, N,N'-dimethyl-4,4'-(biphenyl-2,1-ethenediyl)dipyridinium hexafluorophosphate (BP6). HeLa, Hek293, and Paramecium caudatum cells were stained with BP6. BP6 accumulated on the mitochondria of all three cell types when the mitochondrial membrane potential was high. As the mitochondrial membrane potential decreased following the addition of carbonyl cyanide m-chlorophenyl hydrazine, BP6 moved from the mitochondria to the nucleus in a reversible manner, depending on the mitochondrial membrane potential status. The maximum value of the two-photon absorption cross-section of BP6 is 250 GM (1 GM = 1 × 10(-50) cm(4) s molecules(-1) photon(-1)). This value is 3 and 30 times larger, respectively, than that of the conventional mitochondria selective probes, rhodamine 123 and green fluorescence protein. These results suggest that BP6 should be useful for monitoring mitochondrial membrane potential by two-photon excitation.
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Rab6a releases LIS1 from a dynein idling complex and activates dynein for retrograde movement.
Nat Commun
PUBLISHED: 05-21-2013
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Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules. We previously demonstrated that LIS1 forms an idling complex with dynein, which is transported to the plus ends of microtubules by kinesin motors. Here we report that the small GTPase Rab6a is essential for activation of idling dynein. Immunoprecipitation and microtubule pull-down assays reveal that the GTP bound mutant, Rab6a(Q72L), dissociates LIS1 from a LIS1-dynein complex, activating dynein movement in in vitro microtubule gliding assays. We monitor transient interaction between Rab6a(Q72L) and dynein in vivo using dual-colour fluorescence cross-correlation spectroscopy in dorsal root ganglion (DRG) neurons. Finally, we demonstrate that Rab6a(Q72L) mediates LIS1 release from a LIS1-dynein complex followed by dynein activation through an in vitro single-molecule assay using triple-colour quantum dots. Our findings reveal a surprising function for GTP bound Rab6a as an activator of idling dynein.
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Determination of dissociation constant of the NF?B p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell.
Biochem. Biophys. Res. Commun.
PUBLISHED: 05-21-2013
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Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NF?B dimers in most cells. However, the quantitative value of affinity, namely the K(d), for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K(d) values of mCherry2- and EGFP-fused p50 and p65 were determined to be 0.46 ?M in the cytoplasm and 1.06 ?M in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.
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Siglec-15 Regulates Osteoclast Differentiation by Modulating RANKL-Induced Phosphatidylinositol 3-Kinase/Akt and Erk Pathways in Association With Signaling Adaptor DAP12.
J. Bone Miner. Res.
PUBLISHED: 03-26-2013
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Siglecs are a family of sialic acid-binding immunoglobulin-like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec-15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor ?B ligand (RANKL) signaling in association with DNAX-activating protein 12?kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine-based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec-15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec-15-deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec-15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL-induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt and Erk pathways were impaired in Siglec-15-deficient cells. Retroviral transduction of Siglec-15-null osteoclast precursors with wild-type Siglec-15 or mutant Siglec-15 revealed that the association of Siglec-15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec-15-deficient mice, indicating that there is a compensatory mechanism for Siglec-15-mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast-associated receptor (OSCAR)/Fc receptor common ? (FcR?) signaling, an alternative ITAM-mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec-15-deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec-15-deficient cells when cultured on bone slices, indicating that Siglec-15-mediated signaling can be compensated for by signaling activated by type II collagen and other bone matrix components in the primary spongiosa. Our findings indicate that Siglec-15 plays an important role in physiologic bone remodeling by modulating RANKL signaling, especially in the secondary spongiosa. © 2013 American Society for Bone and Mineral Research.
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Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells.
Mol Biosyst
PUBLISHED: 01-29-2013
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The efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is an important theme in the biomedical and pharmaceutical field. In this study, we synthesized virus-like particles (VLPs) coupled with cyclodextrins (CDs) as hydrophobic pockets through disulfide bonds inside the VLPs, where hydrophobic drugs can be incorporated. We report here the intracellular delivery of hydrophobic dyes or drugs encapsulated in VLPs through CDs with high efficiency and their subsequent release in cells in response to glutathione. As a model anticancer drug, paclitaxel (PTX)-CD complexes were encapsulated inside VLPs and the cytotoxic drug activity of PTX loaded VLPs against NIH3T3 cells was evaluated by CCK-8 assay. PTX-loaded VLPs exhibited a dose-dependent cytotoxic effect with a 20-fold smaller IC(50) than that of free PTX dissolved in DMSO. These results indicate that VLPs with removable CDs afford highly promising carriers of hydrophobic drugs without chemical modification of drugs.
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A Rapid and High-Throughput Quantitation Assay of the Nuclear Factor ?B Activity Using Fluorescence Correlation Spectroscopy in the Setting of Clinical Laboratories.
PLoS ONE
PUBLISHED: 01-01-2013
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Transcription factor nuclear factor-?B (NF-?B) plays a key role in the regulation of immune responses to inflammation. However, convenient assay systems to quantitate the NF-?B activity level in a timely manner are not available in the setting of clinical laboratories. Therefore, we developed a novel and high-throughput quantitative assay based on fluorescence correlation spectroscopy (FCS) to detect the NF-?B activity level in cellular nuclear extracts and evaluated the performance of this method. The basic principle of this assay is to calculate the binding fraction of NF-?B to fluorescent-labeled DNA probes, which contain NF-?B binding sites.
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Fluorescence correlation spectroscopy example: shift of autocorrelation curve.
Cold Spring Harb Protoc
PUBLISHED: 10-05-2011
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Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive tool in biochemistry and biophysics. It directly obtains physical parameters such as the average number of fluorescent molecules and their diffusion time in a tiny detection area. It also provides other useful information such as the brightness of molecules. Ultimately, it can give precise information about molecular interactions in the aqueous condition. In FCS experiments, the fluctuation of fluorescence emission intensity from the tiny detection area is monitored as a function of time. The monitored fluorescence fluctuation signals are transformed to an autocorrelation curve according to the autocorrelation calculator unit and the curves are then fitted to an appropriate physical model. This protocol outlines an FCS example involving a shift of the autocorrelation curve.
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Basic fluorescence correlation spectroscopy setup and measurement.
Cold Spring Harb Protoc
PUBLISHED: 10-05-2011
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Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive tool in biochemistry and biophysics. It directly obtains physical parameters such as the average number of fluorescent molecules and their diffusion time in a tiny detection area. It also provides other useful information such as the brightness of molecules. Ultimately, it can give precise information about molecular interactions in the aqueous condition. This protocol outlines the basic FCS setup and how measurements are made.
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First steps for fluorescence correlation spectroscopy of living cells.
Cold Spring Harb Protoc
PUBLISHED: 10-05-2011
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Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive tool in biochemistry and biophysics. It directly obtains physical parameters such as the average number of fluorescent molecules and their diffusion time in a tiny detection area. It also provides other useful information such as the brightness of molecules. Ultimately, it can give precise information about molecular interactions in the aqueous condition. This article outlines the basic parameters and properties of FCS.
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Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate.
Biochem. Biophys. Res. Commun.
PUBLISHED: 12-05-2010
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Fluorescence cross-correlation spectroscopy (FCCS) reveals information about the spatiotemporal coincidence of two spectrally well-defined fluorescent molecules in a small observation area at the level of single-molecule sensitivity. To simultaneously evaluate the activities of caspase-3 and caspase-9, we constructed a chimeral protein that consisted of tandemly fused enhanced cyan fluorescent protein (ECFP), monomeric red fluorescent protein (mCherry) and monomeric yellow fluorescent protein (Venus). In HeLa cell lysates, a combination of tumor necrosis factor-? (TNF-?)- and cycloheximide (CHX-)-induced apoptosis was monitored. In this, decreases of cross-correlation amplitudes were observed between ECFP and mCherry and between mCherry and Venus. Moreover, time-dependent monitoring of single cells revealed decreases in the cross-correlation amplitudes between ECFP and mCherry and between mCherry and Venus before morphologic changes were observed by laser scanning fluorescence microscopy (LSM). Thus, our method could predict the fate of the cell in the early apoptotic stage.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.