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Kyle R Gee

Publications and source records attributed to Kyle R Gee.

11 recordsLinked to original sources
PubMed2003

Toward the development of new photolabile protecting groups that can rapidly release bioactive compounds upon photolysis with visible light.

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C). The caging group is attached through an ester linkage to the carboxyl functionality of beta-alanine, which activates the inhibitory glycine receptor in the mammalian central nervous system. Such caged compounds play an important role in transient kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged beta-alanine, the caging group is released within 5 micros. Quantum yields of 0.03 and 0.002 were obtained in the UV region (308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments, using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293 cells transiently transfected with cDNA encoding the alpha1 homomeric, wild-type glycine receptor. The experiments demonstrated that neither DANP-caged beta-alanine nor its byproducts inhibit or activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged beta-alanine is water-soluble and stable and can be used for transient kinetic measurements.

Animals↗
PubMed2003

Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events.

Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.

Animals↗
PubMed2003

Modulation of mitochondrial function by endogenous Zn2+ pools.

Recent evidence suggests that intracellular Zn(2+) accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn(2+) that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn(2+) that are mobilized in response to stimulation. One such Zn(2+) pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn(2+) release from one resulting in apparent net Zn(2+) transfer to the other. Further studies found evidence for complex and potent effects of Zn(2+) on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of Deltapsi(m)), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O(2) consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O(2) consumption and increased ROS generation. Finally, strong mobilization of protein-bound Zn(2+) appeared to induce partial loss of Deltapsi(m), suggesting that movement of Zn(2+) between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

Animals↗
PubMed2003

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.

Animals↗
PubMed2003

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye.

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.

Dose-Response Relationship, Drug↗
PubMed2003

A new mitochondrial fluorescent zinc sensor.

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).

Animals↗
PubMed2003

New Ca2+ fluoroionophores based on the BODIPY fluorophore.

Calcium (Ca(2+)) fluoroionophores are useful in cell-based functional assays of G-protein coupled receptor (GPCR) activation or ion channel modulation. In this paper we describe new calcium probes that improve or overcome certain deficiencies in existing probes. These new fluoroionophores are based on acylation of amino-BAPTA [BAPTA = glycine, N,N'-(1,2-ethanediyl-bis(oxy-2,1-phenylene)) bis(N-(carboxymethyl))] with fluorescent BODIPY(R) propionates [BODIPY = 4,4-difluoro-5,7-dimethyl- 4-bora-3a,4a-diaza-s-indacene]. The resulting probes show high affinity to aqueous calcium solutions, and respond to calcium binding with significant fluorescence increases. The BODIPY fluorophores are uncharged and their fluorescence is pH-insensitive. The wide range of excitation/emission wavelength choices available within the BODIPY fluorophore series allows several different colors of new calcium indicators to be prepared. Cell permeable versions respond well with increasing fluorescence intensities in live cells after calcium influx.

Acylation↗
PubMed2003

Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.

Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Mechanism of glutamate receptor-channel function in rat hippocampal neurons investigated using the laser-pulse photolysis (LaPP) technique.

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).

Animals↗
PubMed2002

Detection and imaging of zinc secretion from pancreatic beta-cells using a new fluorescent zinc indicator.

A novel Zn2+-selective visible wavelength fluoroionophore (FluoZin-3, 9) was synthesized. The chelating portion of the molecule resembles known EGTA-based Ca2+-selective fluoroionophores, except that one of the N-acetic acid moieties has been deleted in 9. FluoZin-3 is virtually non-fluorescent in the absence of Zn2+, and exhibits a several hundred-fold fluorescence increase upon saturation with Zn2+( approximately 100 nM), with a Kd = 15 +/- 2 nM. A 1:1 binding stoichiometry of 9:Zn2+ was determined, and the fluorescence of the complex is pH-independent at pH > 6. FluoZin-3 was used to monitor Zn2+ that was co-secreted with insulin from pancreatic beta-cells by exocytosis following stimulation with glucose. The total Zn2+ concentration near the cells reached 600 nM, and Zn2+ was detectable at least 15 mum away from secreting cells. Heterogeneity in secretion among cells was indicated in that some cells in a cluster did not release Zn2+. Also, within secreting cells some regions of the cell membrane gave rise to secretion while others did not, suggesting active zones of secretion on the cell surface.

Cations, Divalent↗