Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.     

Characterization of N- and O-glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI-TOF and quadrupole IT/RTOF mass spectrometry

The structural characterization of the O- and N-glycan structures of three different commercially available recombinant human erythropoietins (rhEPOs) is represented by means of a microscale sample purification using ZipTip technology and MALDI-TOF and MALDI low-energy CID MS. Glycopeptides were released from rhEPO samples by a differential endoproteolytic digestion to obtain site-specific glycosylation patterns. Mass accuracies in the range of +/- 0.04% obtained by the high-resolution TOF instrument allowed an unambiguous assignment of N-glycan structures via glycan database software. Furthermore, the O-glycan structures were directly analyzed on the glycopeptide level by MS/MS experiments. Principally, site-specific glycosylation was found to be very similar for the three different rhEPOs (EPO-alpha, EPO-beta, and novel erythropoiesis stimulating protein (NESP)) but exhibiting quantitative differences in distinct O- and N-glycan moieties. Significant differences were found in the degree of sialylation and acetylation. Especially, a considerable degree of variation of the O-acetylation of sialic acid residues could be realized on the glycan structures of O- and N-glycopeptides, whereas EPO-alpha and EPO-beta could be clearly differentiated from NESP solely on the O-glycopeptide level.     

Development of an iminocoumarin-based zinc sensor suitable for ratiometric fluorescence imaging of neuronal zinc

Ratiometric imaging is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a sensor and is particularly useful for cellular imaging studies. Here we characterized the iminocoumarin fluorophore as a new scaffold for sensors for ratiometric imaging. The iminocoumarin 4 showed a high quantum yield in aqueous media on excitation in the visible wavelength region, while its coumarin analogue showed little fluorescence. We therefore developed a novel fluorescence probe, ZnIC, for ratiometric imaging of Zn2+, using iminocoumarin as a fluorophore and (ethylamino)dipicolylamine as a Zn2+ chelator. ZnIC exhibited almost the same fluorescence properties as 4, and the emission spectrum of this probe was red-shifted on addition of Zn2+ under physiological conditions. ZnIC is selective for Zn2+ over other biologically important metal ions, such as Ca2+ and Mg2+, and has high affinity for Zn2+. To confirm the suitability of ZnIC for biological applications, we employed it for the ratiometric detection of changes in intracellular Zn2+ in cultured cells and in rat hippocampal slices. The results indicate that iminocoumarin is a useful fluorophore for fluorescence microscopic imaging and that ZnIC should be useful for studies on the biological functions of Zn2+.     

Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system

Superior fluorescence imaging methods are needed for detailed studies on biological phenomena, and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy (TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides fluorescence images with nanosecond resolution and has been used to image interactions between proteins, protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent lanthanide complexes (especially europium and terbium trivalent ions (Eu3+ and Tb3+)), in contrast, have long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new luminescent Eu3+ complexes for TRLLM and also developed a new TRLLM system using a conventional fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp as the excitation source. When the newly developed luminescent Eu3+ complexes were applied to living cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was completely excluded. By using Eu3+ and Tb3+ luminescent complexes in combination, time-resolved dual-color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn2+) concentration by using a Zn2+-selective luminescent Eu3+ chemosensor, [Eu-7]. This new imaging technique should facilitate investigations of biological functions with fluorescence microscopy, complementing other fluorescence imaging methodologies.     

Electron dynamics of Cs anti-bonding surface state on copper

Received: 20 September 1998     

Toxicities of dicyanobenzofurazans with formation of superoxide in Escherichia coli

The toxicities of some benzofurazans (BZs), benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), 4,7-dicyanobenzofurazan (5) and 4,5-dicyanobenzofurazan (6), were examined on Escherichia coli. Compound 5 at 4 microM and compound 6 at 7 microM completely inhibited the growth of E. coli in a simple nutritionally restricted medium (GM medium). These compounds were more toxic in GM medium than in a nutritionally rich medium (YE medium), which contained yeast extract as an additive in GM medium. Compound 4 also inhibited the growth of E. coli at 300 microM in GM medium. The toxicities of BZs were in the order of 1 approximately 2 approximately 3 less than 4 approximately 5 approximately 6. Compounds 4, 5 and 6 induced manganese-superoxide dismutase (Mn-SOD) and catalase activities of E. coli in YE medium. The induced SOD and catalase provide a defense against the potential cytotoxicities of O2- and H2O2. The rate of dioxygen uptake in cyanide-resistant respiration of E. coli was dependent on the concentration of 5, and was correlated with the induction of SOD and catalase. The reduction potentials of BZs followed the order of 1 approximately 2 less than 3 less than 4 less than 5 approximately 6. Compounds 5 and 6, which had redox potentials higher than those of the other BZs, are thought to be more readily reduced in the living system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of fluorescein as a platform for finely tunable fluorescence probes

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.     

Parallel excitation pathways in ultrafast interferometric pump-probe correlation measurements of hot-electron lifetimes in metals

Hot electron (E-E_Fermi=0.75 to 1.55 eV) lifetimes for cesiated Cu(100) and Cu(111) surfaces are measured via interferometric time-resolved two-photon photoemission with a 19-fs intensity FWHM mode locked Ti:sapphire laser at 1.55 eV. The data are analyzed using the optical Bloch equations and a laser pulse characterized in situ via surface second-harmonic generation interferometric autocorrelation. It is found that the retrieved hot-electron lifetimes are unphysically fast, and have a strong dependence on the temperature of the sample and the polarization of the laser. A simple explanation for the data is that the measured signal consists of contributions from transitions through both virtual and real intermediate states. Received: 26 July 2000 / Accepted: 8 September 2000 / Published online: 12 October 2000     

Micro-combustion Calorimetry Aiming At 1 mg Samples

A micro-combustion calorimeter was developed. The small energy equivalent (ca. 68 JK_–1) of this calorimeter makes it possible to measure combustion energies of very small samples. The energy equivalent was determined by burning 2 mg of benzoic acid. The standard deviation of the mean energy equivalent was reduced to 0.014% in 5 experiments. The standard massic energy of combustion of salicylic acid and the standard deviation of the mean were determined to be –21871±5 J g_–1, which agrees well with the literature values. The standard molar enthalpy of formation of salicylic acid was derived as –591.2±1.7 kJ mol_–1.This revised version was published online in November 2005 with corrections to the Cover Date.