Study of the interaction between actin and antitumor substance aplyronine A with a novel fluorescent photoaffinity probe

The interaction between actin and aplyronine A, a potent antitumor and actin-depolymerizing substance of marine origin, was investigated by photoaffinity labeling experiments. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophore as a detecting group were synthesized. Photolabeling experiments between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chemical evidence for the direct interaction between actin and the side-chain portion of aplyronine A.     

A novel adaptive fluorescent probe for cell labelling

In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy.     

A novel fluorescent probe for protein binding and folding studies: p-cyano-phenylalanine

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (PheCN), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using PheCN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single PheCN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the PheCN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (Kd) of this peptide to CaM. Taken together, these results indicated that the PheCN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.     

A novel fluorescent probe: europium complex hybridized T7 phage

We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.     

Synthesis of a novel fluorescent probe for estrogen receptor

A novel estradiol-mimetic fluorescent probe 5 was synthesized from diethylstilbestrol (DES, 1), which is useful for probing estrogen receptor (ERalpha), a prognostic indicator of estrogen-dependent cancers, and for developing a homogeneous fluorescence polarization (FP) assay to identify the ligands of estrogen receptor.     

Inorganic phosphate nanorods are a novel fluorescent label in cell biology

We report the first use of inorganic fluorescent lanthanide (europium and terbium) ortho phosphate [LnPO₄·H₂O, Ln = Eu and Tb] nanorods as a novel fluorescent label in cell biology. These nanorods, synthesized by the microwave technique, retain their fluorescent properties after internalization into human umbilical vein endothelial cells (HUVEC), 786-O cells, or renal carcinoma cells (RCC). The cellular internalization of these nanorods and their fluorescence properties were characterized by fluorescence spectroscopy (FS), differential interference contrast (DIC) microscopy, confocal microscopy, and transmission electron microscopy (TEM). At concentrations up to 50 μg/ml, the use of [³H]-thymidine incorporation assays, apoptosis assays (TUNEL), and trypan blue exclusion illustrated the non-toxic nature of these nanorods, a major advantage over traditional organic dyes     

A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258, our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT assays.     

Synthesis of a novel fluorescent probe useful for DNA detection

In this paper, a novel fluorescent probe 2-methylbenzo[b][1,10] phenanthrolin-7(12H)-one (m-BPO) is synthesized, and its molecular structure has been characterized by IR, UV, MS, (1)H-NMR and elements analysis. The fluorescent characteristics of m-BPO were investigated in detail. It was found that DNA had the ability to quench the fluorescence of m-BPO at 411 nm (lambda(ex)=286 nm), and the quenched intensity of fluorescence was proportional to the concentration of DNA. Based on this fact, m-BPO has been used as the fluorescent probe for detection of calf thymus DNA (ctDNA) and fish semen DNA (fsDNA). Under the optimal conditions, the calibration curves are linear up to 15.0 microg/ml for both ctDNA and fsDNA. The corresponding detection limits are 3.6 ng/ml for ctDNA and 5.5 ng/ml for fsDNA, respectively. The interaction mechanism for the binding of m-BPO to ctDNA was studied in detail, and the results suggested that the interaction mode between m-BPO and ctDNA was groove binding.     

7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin: Synthesis of a fluorescent actin probe

Phallacidin was labeled with the fluorophore, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, after a series of modifications. The complete purification and synthesis of intermediates are described. A simple laboratory procedure for production of the fluorescent toxin is outlined. The fluorescent phallacidin was used to stain plant and animal cellular actin. Because of the low molecular weight, high actin specificity and visible excitation profile of the conjugated toxin, fluorescent phallacidin, has proven useful in the fluorescence microscopic study of actin in vivo and in vitro.     

Synthesis and characterization of a novel near-infrared fluorescent probe for applications in imaging A549 cells

Objective

To design and synthesize a novel near-infrared (NIR) fluorescent probe based on indocyanine Green (ICG), that can be applied in imaging living cells.

Results

A highly fluorescent novel NIR fluorescent probe (IR-793) was synthesized in two steps. IR-793 had better fluorescence and optical stability than ICG. In addition, no obvious cytotoxicity effect of IR-793 was observed and cell viability was above 75% at the maximum concentration (120 nM). IR-793 also exhibited good performance in imaging living A549 cells.

Conclusion

IR-793, a novel NIR fluorescent probe that is stable, low-cost, highly fluorescent and low cytotoxicity, has been designed and synthesized for imaging living cells.

     

A novel colorimetric fluorescent probe for detecting hydrazine in living cells and zebrafish

Hydrazine (NH₂NH₂) is a highly toxic organic substance that poses a threat to human health. Monitoring hydrazine with high sensitivity and selectivity is very important. Here, a simple colorimetric fluorescent probe for hydrazine detection, which is a seminaphthorhodafluor derivative containing thiophene-2-carboxylic acid ester reaction site, was rationally constructed. The probe itself exhibits weak fluorescence. The fluorescence is significantly enhanced when hydrazine is added. The probe exhibited a broad linear range (0–1 mM) with satisfactory selectivity and sensitivity (limit of detection 36.4 μM), which turned out to be an excellent fluorescent probe for monitoring hydrazine. Additionally, the probe was used to track hydrazine in living cells and zebrafish with great success, and the detection performance was satisfying. These results proved that this type of fluorescent probe with the thiophene-2-carboxylic acid ester structure can detect hydrazine with higher selectivity and sensitivity.     

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1 . LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.     

A fast‐responsive fluorescent probe for sulfite and its bioimaging

A turn‐on fluorescent probe Coumarin‐SO2 based on a nucleophilic addition reaction was developed for the rapid detection of SO₃^2– in aqueous media. The probe Coumarin‐SO2 displays excellent water solubility, fast response, highly sensitivity and highly selectivity over other biological related species. More importantly, living cell imaging experiments indicate the feasibility of using the probe for the detection of SO₃^2– in biological systems. Copyright © 2015 John Wiley & Sons, Ltd.     

Orientation of actin monomer in the F-actin filament: radial coordinate of glutamine-41 and effect of myosin subfragment 1 binding on the monomer orientation

We have employed the method of radial distance measurements in order to orient the actin monomer in the F-actin filament. This method utilizes fluorescence resonance energy transfer measurements of the distance between two equivalent chemical points located on two different monomers. The interprobe distance obtained this way is used to compute the radial coordinate of the labeled amino acid [Taylor, D. L., Reidler, J., Spudich, J. A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367]. Theoretical analysis has indicated that if radial coordinates of four points are determined and six intramolecular distances are known, one can, within symmetry limits, position the monomer about the filament axis. The radial distance of Gln-41 that had been enzymatically modified with dansyl, rhodamine, and fluorescein derivatives of cadaverine was found to be approximately 40-42 A. The determination of the radial distance of Cys-374 was accomplished by using monobromobimane and N-[[(iodoacetyl)amino]ethyl]-5- naphthylamine-1-sulfonate as donors and N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide as acceptor; the results were consistent with a radial coordinate for this residue of 20-25 A. The effect of myosin subfragment 1 (S1) binding on the radial coordinates of (1) Gln-41, (2) Cys-374, and (3) the nucleotide binding site was also examined. S1 had a small effect on the radial coordinate of Gln-41, increasing it to 44-47 A. In the two remaining lases the change in the radial coordinate due to the S1 binding was negligible. This finding excludes certain models of the interaction between actin and S1 in which actin monomer rotates by a large angle when subfragment 1 binds to it.     

Study of actin and its interactions with heavy meromyosin and the regulatory proteins by the pulse fluorimetry in polarized light of a fluorescent probe attached to an actin cysteine.

The decay of anisotropy of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine fluorescence attached to cysteine-373 of actin can be characterized by two correlation times theta1 and theta2. theta1 has a value of several nanoseconds and is thought to represent some local protein motion. theta2 is of the order of several hundreds of nanoseconds. Its value increases with actin concentration. It represents an average of the G and F actin correlation times. When actin interacts with heavy meromyosin, theta2 increases and becomes infinite at a molar ratio of one heavy meromyosin molecule per four actin protomers. It is concluded that a definite complex is then formed between F actin and heavy meromyosin. In the same time, G actin concentration becomes equal to zero. Finally, when F actin forms a complex with the regulatory proteins tropomyosin and troponin, the value of theta2 is greater in the absence than in the presence of Ca2+. This result indicates that micromolar concentrations of Ca2+ induces a conformation change of the complex of F actin with the regulatory proteins.     

A novel fluorescent probe that is brain permeable and selectively binds to myelin.

Myelin is a multilayered glial cell membrane that forms segmented sheaths around large-caliber axons of both the central nervous system (CNS) and peripheral nervous system (PNS). Myelin covering insures rapid and efficient transmission of nerve impulses. Direct visual assessment of local changes of myelin content in vivo could greatly facilitate diagnosis and therapeutic treatments of myelin-related diseases. Current histologic probes for the visualization of myelin are based on antibodies or charged histochemical reagents that do not enter the brain. We have developed a series of chemical compounds including (E,E)-1,4-bis(4'-aminostyryl)-2-dimethoxy-benzene termed BDB and the subject of this report, which readily penetrates the blood-brain barrier and selectively binds to the myelin sheath in brain. BDB selectively stains intact myelinated regions in wild-type mouse brain, which allows for delineation of cuprizone-induced demyelinating lesions in mouse brain. BDB can be injected IV into the brain and selectively detect demyelinating lesions in cuprizone-treated mice in situ. These studies justified further investigation of BDB as a potential myelin-imaging probe to monitor myelin pathology in vivo.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

A novel nitro-substituted benzothiadiazole as fluorescent probe for tumor cells under hypoxic condition

Most of solid tumor cells are hypoxic and hard to trace and measure. A new compound, 4,7-bis(4-dodecylthiophen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO₂), was synthesized for labeling the hypoxic cells specially in this paper. BTTD-NO₂ showed no cytotoxicity to MG63 cells by MTT method. When MG63 cells were cultured with BTTD-NO₂ under hypoxic condition for 24 h, strong red fluorescence distribution in cytoplasm was observed. Flow cytometry results showed that 65% of MG63 cells were labeled with strong red fluorescence in hypoxic condition while only 2.4% in oxic condition. Furthermore, Real time RT-PCR proved that BTTD-NO₂ could stimulate high gene expression of the nitroreductase in the cells which could improve the conversion rate of BTTD-NO₂ to BTTD-NH₂ in turn. It proved that the fluorescence of BTTD-NO₂ was quenched by its two nitro groups, however, strong red fluorescence could emit in the cytoplasm after the reduction of its nitro groups to amino groups in the tumor cells under hypoxic condition. These results suggested that BTTD-NO₂ had the potential as a superior fluorescent probe for tumor detection.