Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

Furuta, Toshiaki; Takeuchi, Hiroko; Isozaki, M.; Takahashi, Yasuhiro; Kanehara, Makoto; Sugimoto, Motokazu; Watanabe, Tomohiro; Noguchi, Kousei; Dore, Timothy M.; Kurahashi, Takashi; Iwamura, Michiko; Tsien, Roger Y.

doi:10.1002/cbic.200300814

Cited by 98 publications

(79 citation statements)

References 42 publications

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“…Because canaliculi are contractile structures, the function of which is governed by cytoskeletal proteins and actin filaments in the pericanalicular region of hepatocytes, it is possible the canalicular changes observed may be attributable to ANIT induced (either direct or indirect) cytoskeletal alterations in hepatocytes. Cytoskeletal derangements resulting from ANIT exposure have been observed in isolated rat hepatocyte couplets (Orsler et al 1999), and in vivo (Furuta et al 2004;Kan and Coleman 1986;Lowe et al 1985). Hence, mammalian studies suggest ANIT induced cytoskeletal alterations a plausible mechanism by which the observed canalicular attenuation/dilation in medaka may manifest.…”

Section: Canalicular Attenuation and Dilationmentioning

confidence: 98%

Non invasive high resolution in vivo imaging of α-naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity in STII medaka

et al. 2008

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A novel transparent stock of medaka (Oryzias latipes; STII), homozygous recessive for all four pigments (iridophores, xanthophores, leucophores, melanophores), permits transcutaneous, high resolution ( < 1μm) imaging of internal organs and tissues in living individuals. We applied this model to in vivo investigation of α-napthylisothiocyanate (ANIT) induced hepatobiliary toxicity. Distinct phenotypic responses to ANIT involving all aspects of intrahepatic biliary passageways (IHBPs), particularly bile preductular epithelial cells (BPDECs), associated with transitional passageways between canaliculi and bile ductules, were observed. Alterations included: attenuation/ dilation of bile canaliculi, bile preductular lesions, hydropic vacuolation of hepatocytes and BPDECs, mild BPDEC hypertrophy, and biliary epithelial cell (BEC) hyperplasia. Ex vivo histological, immunohistochemical, and ultrastructural studies were employed to aid in interpretation of, and verify, in vivo findings. 3D reconstructions from in vivo investigations provided quantitative morphometric and volumetric evaluation of ANIT exposed and untreated livers. The findings presented show for the first time in vivo evaluation of toxicity in the STII medaka hepatobiliary system, and, in conjunction with prior in vivo work characterizing normalcy, advance our comparative understanding of this lower vertebrate hepatobiliary system and its response to toxic insult.

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Section: Canalicular Attenuation and Dilationmentioning

confidence: 98%

Non invasive high resolution in vivo imaging of α-naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity in STII medaka

et al. 2008

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“…NPE-IP 3 has a low two-photon crosssection (|[sim]| 0.001 GM), so a caged IP 3 with larger cross-section (0.035 GM) has been synthesized in seven synthetic steps, permitting localized two-photon uncaging of IP 3 in living cells 37 . Finally, membrane-permeant ester derivatives of caged cAMP and IP 3 probes have been made using multistep syntheses that permit loading of these second messengers into intact cells 38 , 39 , 40 .…”

Section: Caged Nucleotides Nucleosides and Inositolsmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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“…Initially, a DAG-γ-lactone template was prepared as reported previously. 21) Oxidation of 7-diethylamino-4-methylcoumarin with selenium dioxide gave DEAC possessing an aldehyde group at the C4 position, 22) and this was followed by an aldol condensation with the lactone 3. Deprotection of tertbutyldiphenylsilyl (TBDPS) and finally, monoacylation of the resulting diol gave sn-2 DEAC-type DAG-lactone 6 (E isomer, racemic) (Chart 1).…”

Section: )mentioning

confidence: 99%

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

Ohashi

Nomura

Minato

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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Protein kinase C (PKC) is correlated with cell signaling pathways and also receives attention as a therapeutic target for cancer and Alzheimer-type dementia. The application of Förster/fluorescence resonance energy transfer (FRET) phenomena to detect binding between proteins and small molecules, for example, PKC and its ligands, underlies a fluorescence-based assay method suitable for high-throughput screening. To accelerate studies on PKC functions in processing signals using small molecules and the development of drugs that target PKC, novel methods for the assessment of the PKC binding affinity of compounds are necessary. We previously developed solvatochromic fluorophore-based methods for that assessment. In this study, a novel method for a FRET-based PKC binding assay was developed and is expected to overcome the limitations of solvatochromic fluorophores.Key words protein kinase C; protein kinase C ligand; ligand screening; Förster resonance energy transfer; fluorescence resonance energy transfer Förster/fluorescence resonance energy transfer (FRET) is the transfer of excitation energy from a fluorescence donor to an acceptor. FRET efficiency and sensitivity depend on the distance between the donor and the acceptor. The FRET phenomenon can be applied as a spectroscopic measure and a probe of conformational change of macro-biomolecules [1][2][3] and a FRET-mediated competitive assay can be used for studies of the binding between two molecules. 4,5) Radioisotopebased methods are extremely sensitive and are widely used for ligand binding assays, but fluorescent-based assays, which are suitable for high-throughput screening are free of hazards associated with radioactivity. Previously, we described fluorescence-based ligand screening assays as an alternative to radioisotope assays. 6,7)Protein kinase C (PKC) isozymes, which are classified as Ser/Thr kinases, play a critical role in cellular signaling pathways related to proliferation, 8,9) differentiation, 10) and apoptosis.11,12) PKC has 11 isozymes which are classified into three subtypes: conventional PKC (cPKC; α, β I/II , γ), novel PKC (nPKC; δ, ε, η, τ) and atypical PKC (aPKC; ζ, λ, ι). The binding of diacylglycerols (DAG) to the C1 domain of PKC is an important step in an activation process except in the case of aPKC which contains an atypical C1 domain which fails to bind to DAG.13) Various synthetic PKC ligands targeting the C1 domain have been developed as chemical probes or drug candidates by radioisotope assays.14-17) We have previously developed a fluorescent assay for PKC-ligand binding affinity using a synthetic PKC δC1b domain labeled with a solvatochromic fluorophore on the edge of its ligand-binding pocket. This can detect ligand binding through changes in the surrounding environment.18) However, this method would not be suitable for high-throughput screening. The reason might be due to several possibilities: For instance, the change of fluorescence intensity is affected by properties of test compounds. It would be concerned that a fluorescen...

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Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

Cited by 98 publications

References 42 publications

Non invasive high resolution in vivo imaging of α-naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity in STII medaka

Non invasive high resolution in vivo imaging of α-naphthylisothiocyanate (ANIT) induced hepatobiliary toxicity in STII medaka

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

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