Bioluminescence color modulation of Vibrio fischeri strain Y1 coupled with alterable levels of endogenous yellow fluorescent protein and its fluorescence imaging

Bioluminescence (BL) (lambda(max) approximately 535 nm) of Vibrio fischeri strain Y1 has been previously characterized in terms of the fluctuation in intracellular levels of yellow fluorescent protein (YFP). In this study fluorescence microscopic analysis has revealed that yellow fluorescence, as well as blue fluorescence attributable to a luciferase intermediate, is localized to the periphery of V. fischeri Y1 cells. This finding indicates that both YFP and the luciferase are present in the vicinity of the cell membrane. By using cyanide to enhance yellow BL, it has been shown that BL modulation is coupled with the fluctuations in the intracellular levels of YFP and the primary emitter. On the basis of the BL characterization, combined with results of a sedimentation experiment, it has been shown that larger cells produce a relatively stronger yellow BL. Two-dimensional gel electrophoresis of cell-protein extracts has shown that the YFP level is more alterable than the luciferase level. It is postulated that the yellow BL modulation takes place in connection with cell growth.     

A BLUE FLUORESCENT PROTEIN FROM A YELLOW-EMITTING LUMINOUS BACTERIUM

Vibrio fischeri strain Y1 emits yellow light in vivo due to the participation of a yellow fluorescent protein (YFP) in the luciferase reaction. In this study it was found that the organism also produces a protein (referred to as Y1-BFP) emitting strong blue fluorescence. Its molecular weight, about 25 kDa, is the same as or very close to that of YFP. The fluorescence excitation and emission maxima of the purified Y1-BFP are at 416 and 461 nm, respectively, and the fluorescence lifetime is 12.5 ns at 2 degrees C. The molar extinction coefficient of Y1-BFP at 416 nm was estimated to be approx. 9500. With the homologous luciferase, Y1-BFP decreases the intensity and rate of decay in the in vitro reaction but has no effect on its emission spectrum (in contrast to YFP, which has a striking effect on the spectrum). With luciferase isolated from Vibrio harveyi, however, Y1-BFP causes a small blue-shift (approximately 10 nm) in the emission of the enzyme catalyzed reaction, whereas YFP has no effect on the emission spectrum.     

Effectiveness of the Accessory Yellow Fluorescent Protein in the Bacterial Luciferase Reaction Correlates with the Lifetime of the Peroxyhemiacetal Intermediate: The Stereochemistry of the Reaction

In the in vitro reaction of Vibrio fischeri Y-1 luciferase, the dependence of the initial luminescence intensity (I_o) and its rate of decay (k_d) on the chain-length of the aliphatic aldehyde are greatly altered by the presence of yellow fluorescent protein (YFP), which functions as an accessory emitter. Whereas with no YFP both k_d and I_o are maximum for chain lengths ≥ 12, the fraction of the light emitted from the accessory chromophore, measured as the ratio of yellow to blue light (Y/B), is greater with shorter chain-length aldehydes. Thus, aldehydes that are least efficient in the absence of YFP are more efficient for causing yellow emission in its presence. These results are interpreted on the basis of the expected lifetimes of the peroxyhem-iacetals with which YFP interacts: high values of k_d reflect short peroxyhemiacetal lifetimes, hence less chance of interaction with YFP. The critical dependence on aldehyde chain-length underlines the importance of stereochemical factors in the bacterial reaction, which are discussed here in the framework of a chemically initiated electron exchange luminescence model.     

Photochemical properties and sensor applications of modified yellow fluorescent protein (YFP) covalently attached to the surfaces of etched optical fibers (EOFs)

Fluorescent proteins have the inherent ability to act as sensing components which function both in vitro and inside living cells. We describe here a novel study on a covalent site-specific bonding of fluorescent proteins to form self-assembled monolayers (SAMs) on the surface of etched optical fibers (EOFs). Deposition of fluorescent proteins on EOFs gives the opportunity to increase the interaction of guided light with deposited molecules relative to plane glass surfaces. The EOF modification is carried out by surface activation using 3-aminopropylthrimethoxysilane (APTMS) and bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) which exposes sulfhydryl-reactive maleimide groups followed by covalent site-specific coupling of modified yellow fluorescent protein (YFP). Steady-state and fluorescence lifetime measurements confirm the formation of SAM. The sensor applications of YPF SAMs on EOF are demonstrated by the gradual increase of emission intensity upon addition of Ca²⁺ ions in the concentration range from a few tens of micromolars up to a few tens of millimolars. The studies on the effect of pH, divalent cations, denaturing agents, and proteases reveal the stability of YFP on EOFs at normal physiological conditions. However, treatments with 0.5% SDS at pH 8.5 and protease trypsin are found to denaturate or cleave the YFP from fiber surfaces.     

AUTOINDUCTION AND ALDEHYDE CHAIN-LENGTH EFFECTS ON THE BIOLUMINESCENT EMISSION FROM THE YELLOW PROTEIN ASSOCIATED WITH LUCIFERASE IN Vibrio fischeri STRAIN Y-1b

Abstract— A new strain of the yellow-light emitting bacterium Vibrio fischeri Y-lb, possessing a higher fraction of its emission in the yellow, was isolated and characterized. The in vivo yellow to blue ratio (Y/B) increases during growth in parallel with the autoinduction of luciferase, reaching a value above 5 at 17°C. This Y/B increase is attributed to concomitant increases in the intracellular levels of both luciferase and the specific protein associated with yellow emission, the yellow fluorescent protein (YFP), and to the association of the two proteins at higher concentrations. The yellow emission is rapidly but reversibly lost by brief exposures to a temperature of 27°C (5 s), and irreversibly so at 40°C, attributed to dissociation of the two proteins. A concentrated crude extract emits almost exclusively yellow light in the in vitro reaction, and theY/B ratio decreases with dilution. The Y/B ratio in vitro is also dependent on the aldehyde chain length; experiments with aldehyde analogs suggest that the aldehyde binding site of luciferase must be occupied for YFP to stimulate light emission.     

Triple channel sensing of Pb(II) ions by a simple multiresponsive ferrocene receptor having a 1-deazapurine backbone

A new redox, chromogenic, and fluorescent chemosensor molecule based on a deazapurine ring selectively senses aqueous Pb2+ in acetonitrile over other metal ions examined: redox shift (DeltaE1/2 = 0.15 V of the Fe(II)/Fe(III) redox couple), the colorless to orange color change, and an emission change of 620-fold, with an unprecedented detection limit of 2.7 microg L-1. The signal transduction occurs via a reversible CHEF with this inherent quenching metal ion.     

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.     

Genetically encoded fluorescent reporters of histone methylation in living cells

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.     

Pentadecatungstate with dinuclear cerium(III) unit: synthesis, crystal structure and properties

A novel pentadecatungstate, [H 6Ce 2(H 2O)Cl(W 5O 18) 3] (7-) ( 1), constructed by a dinuclear cerium unit and 15-member ring WO 6 units was prepared and characterized by single-crystal X-ray diffraction. Polyanion 1 exhibits blue photoluminescence with an emission maximum at 488 nm, which is characteristic of cerium(III) transitions from 5d to (2)F 5/2 states. Furthermore, the study of the electrochemical property investigation of 1 shows two reversible redox peaks ascribing to two-electron processes.     

Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface-accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide-thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state.     

A simple and robust reversible redox-fluorescence molecular switch based on a 1,4-disubstituted azine with ferrocene and pyrene units

Taking advantage of the properties of the ferrocene as a redox and electron donor active unit and the pyrene as a fluorescent unit, dyad 2 shows a fast and reversible redox-switchable fluorescence emission.     

Detection of mitochondrial caspase activity in real time in situ in live cells.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP-caspase 3 substrate-YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.     

Reversible electrochemical conversion between Rh(II) and Rh(III) states in Rh porphyrin adsorbed on carbon black

We have clearly demonstrated reversible cyclic voltammograms for the redox reaction between the Rh(II) and Rh(III) states in rhodium octaethylporphyrin [Rh(OEP)] adsorbed on carbon black in an acidic aqueous solution. The emergence of the reversible wave can be ascribed to the suppression of the undesirable reactions between two molecules of [Rh(II)(OEP)] because of its strong interaction with the carbon black. The generated [Rh(II)(OEP)] exhibits a potent catalytic O(2) reduction activity.     

CH_4/CO_2重整制合成气Ni/Ce_xZr_(1-x)O_2催化剂性能研究

以水热法合成的介孔铈锆固溶体为载体,采用浸渍法制备了Ni/CexZr1-xO2催化剂,使用X射线衍射(XRD)、程序升温还原(TPR)和热重-差示扫描量热分析(TG-DSC)等测试手段对催化剂进行了表征。通过对比以ZrO2,CeO2,Ce0.6Zr0.4O2和Ce0.33Zr0.67O2为载体的Ni基催化剂性能,发现铈锆固溶体独特的氧化还原性质可以提高活性组分的分散度,增强催化剂的抗积炭性能,从而提高催化剂对甲烷二氧化碳重整制合成气的选择性。     

Study the oxidative injury of yeast cells by NADH autofluorescence

Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H(2)O(2) and ONOO(-) have been recorded in detail in this work. In the presence of different amounts of H(2)O(2) and ONOO(-), necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.     

Three-point recognition and selective fluorescence sensing of L-DOPA

[reaction: see text] A phenylboronic acid derivative of a well-known dye (Lucifer yellow) recognizes L-DOPA through a combination of reversible esterification, charge transfer, and electrostatic interactions. The selective recognition event is signaled by a drop in the emission intensity of the fluorescent chemosensor.     

The photoyellowing of stilbene-derived fluorescent whitening agents--mass spectrometric characterization of yellow photoproducts.

The application of fluorescent whitening agents (FWAs) significantly accelerates the photoyellowing of wool and silk under exposure to the ultraviolet and visible components of sunlight <500 nm. The photochemistry involved in this process is poorly understood, particularly the role of photoproducts derived directly from the FWA itself. Hydroxylation was identified as the key initial mechanism of photodegradation leading to coloration of the solution in the irradiation of the stilbene-derived FWA 4,4'-bis(2-sulfostyryl)biphenyl (DSBP) in the presence of hydrogen peroxide (H2O2). Polyhydroxylated DSBP derivatives were implicated as critical intermediates in the formation of yellow photoproducts under these conditions. The formation of trace quantities of DSBP quinone derivatives subsequent to hydroxylation was identified as the key cause of DSBP photoyellowing. These results are the first successful characterization of yellow photoproducts resulting directly from irradiation of a stilbene-based FWA. Formation of these yellow stilbene-based FWA-derived photoproducts may occur on the surface of FWA-treated wool exposed to simulated sunlight, as previous work has shown that H2O2 is photogenerated when wet FWA-treated wool is exposed to light. These results therefore suggest that yellow FWA-derived photoproducts contribute to the accelerated photoyellowing of FWA-treated wool.     

Engineering strain-sensitive yellow fluorescent protein

Various biological events including muscle contraction and vesicle transport can be described as a mechanical process. Many of the corresponding proteins are thus required to generate or sense a force. Here we describe a strain-sensitive yellow fluorescent protein (YFP) recombinant that can detect the intramolecular strains these proteins experience.     

A novel genetically encoded fluorescent protein as a Cu(I) indicator

We constructed the copper(I)-binding domain of Mycobacterium tuberculosis (Mtb) CDC 1551 (residues 1-162) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), and formed a novel genetically encoded fluorescent copper(I) responsive protein (PMtb). The sensitivity and selectivity to copper(I) of the PMtb was sought. The experiments showed that the copper(I)-binding domain of the PMtb was highly sensitive and selective towards copper(I).     

A highly selective fluorescent chemosensor for Pb2+

A new fluorescent sensor based on rhodamine B for Pb2+ was synthesized. The new fluorescent sensor showed an extreme selectivity for Pb2+ over other metal ions examined in acetonitrile. Upon the addition of Pb2+, an overall emission change of 100-fold was observed, and the selectivity was calculated to be 200 times that of Zn2+. The signal transduction occurs via of reversible CHEF (chelation-enhanced fluorescence) with this inherent quenching metal ion.