Kyle Gee

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is a recently identified metabolite of NADP+ that is as potent as inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca2+ release activated by NAADP+ and the Ca2+ stores it acts on are different from those of IP3 and cADPR. In this study we show that photolyzing caged NAADP+ in intact sea urchin eggs elicits long term Ca2+ oscillations. On the other hand, uncaging threshold amounts of NAADP+ produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP+ receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP+ can fully inactivate subsequent binding to the receptor in a time-dependent manner. Thus, the NAADP+-sensitive Ca2+ release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca2+ mobilization.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Caged compounds are molecules whose biological function is masked until UV light induces a photo-chemical reaction that converts the molecules into a biologically active state. These probes provide very powerful tools in cell biology research, since the activation can be precisely controlled temporally and spatially by limiting their exposure to light. This report describes a simple caged compound illuminator that can be attached to an inverted microscope; it can simultaneously provide both sufficient UV illumination for photoactivation and epifluorescence excitation to monitor indicator fluorescence. As an example, we show that the cytoplasmic calcium level can be raised very rapidly by the photoactivation of a new caged compound, caged calcium ionophore DMNPE-A23187, and that this [Ca2+]i increase is associated with tail retraction of motile keratocytes.

A series of novel sodium ion-sensitive fluorescent reagents suitable for biological applications is described. The chelator nitrogen atom substituents affect the selectivity and affinity of cation binding, while the nature of the fluorophore determines the type of fluorescent response to metal ion chelation.

We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profiles of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae. The PBP profiles obtained are virtually identical to those reported previously with 3H-, 14C-, or 125I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for di...

Newly synthesized photolabile derivatives of glutamate, caged glutamate, that release free glutamate on a microsecond time scale after a pulse of UV laser light are described. 2-Nitrobenzyl derivatives were attached to the amino or carboxyl groups of glutamate. Substitution with a -CO2- group at the benzylic carbon accelerates the photolysis reaction when compared to -H and -CH3 substituents. gamma-O-(alpha-Carboxy-2-nitrobenzyl)glutamate is stable at neutral pH. In 100 mM phosphate buffer at pH 7.0, the compound is photolyzed at 308 nm with a quantum product yield of 0.14. The half-life of the major component of the photolytic reaction, as judged by the transient absorbance change at 430 nm, is 21 microseconds (approximately 90%); the half-life of a minor component (approximately 10%) is 0.2 ms. The amino-linked derivatives have half-lives in the millisecond region and a 4-fold lower quantum yield. The potential of the newly synthesized compound for use in rapid chemical kinetic in...

The stepwise ionization (H(3)R(+) <==> H(2)R <==> HR(-) <==> R(2-)) of four fluorescein fluoro derivatives was studied by visible spectroscopy. The pK(a) values were determined in water, in 50 mass % aqueous ethanol, in oil-in-water microemulsions (benzene + CTAB + pentanol-1 in water with 1.0 M KCl; CTAB = cetyltrimethylammonium bromide), and in reversed ones (water + AOT in n-octane; AOT = bis-2-ethylhexylsulphosuccinate or Aerosol OT). The medium effects, DeltapK(a), i.e., changes in pK(a) of these dyes on going from water to some other solvent systems, were rationalized by considering the tautomerism, the values of microscopic ionization constants, and the charge types of the acid-base couples. An expressed shift of the tautomeric equilibria of H(2)R toward colorless lactone was registered on going from water to both aqueous ethanol and organized solutions. While the monoanions HR(-) of 3',4',5',6'-tetrafluoro- and 2,7,3',4',5',6'-hexafluorofluorescein exist in all the systems studied as a tautomer with ionized carboxylic and nonionized hydroxy groups, in the case of 2,4,5,7-tetrafluorofluorescein, the prevalence of another tautomer was observed (COOH and O(-) groups). For 2,7-difluorofluorescein (Oregon Green 488), the partial shift of the tautomeric equilibrium of HR(-) was registered from (COO(-) and OH) in water to (COOH and O(-)) in other solvent systems. The data for the dyes located in an AOT-based pseudophase indicate that the interior of the latter exerts essential differentiation of the acid strength of the dyes, probably caused by the peculiarity of dye species location in water pools. While the state of tautomeric equilibria resembles that in nonaqueous media, the absorption maxima of R(2-) species are close to those in water. Such nonuniform influence displayed by AOT-based water droplets should be taken into account when examining them by using different molecular probes.

Using in vitro fast scan cyclic voltammetry, we measured cocaine potency for inhibiting dopamine uptake/clearance in accumbens slices 7 days after withdrawal from chronic cocaine pretreatments. Rats were pretreated with 40 mg/kg per day for 14 days, either via continuous osmotic minipumps or by once-daily injections. The cocaine potency was subsequently assessed for endogenous and exogenous dopamine applied via single-pulse electrical stimulation and caged-dopamine photolysis, respectively. Under baseline conditions, no differences in either endogenous or exogenous dopamine kinetics were observed in the two cocaine pretreatment groups. In contrast, the potency of bath-applied cocaine for inhibiting endogenous dopamine uptake was enhanced in the intermittent injection group with no change in the continuous infusion group. The selective increase in the cocaine potency following injections was also demonstrable for clearance of photo-applied DA. The enhanced cocaine potency in the accumbens slices following 7 days of withdrawal is consistent with the residual sensitization to cocaine-induced locomotion following daily cocaine injections. Behavioral tolerance following continuous infusion, on the other hand, may be mediated via a mechanism distinct from altered dopamine uptake.

Rapid release of biologically active molecules from photolabile, or" caged", precursors is an important technique in the study of fast biological processes.'Photolysis of caged compounds provides temporal and spatial control of the distribution of substrate ...

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.

The application of the desyl or 2-oxo-1,2-diphenylethyl moiety as a photolabile ligand for the release of phosphates such as cAMP and inorganic phosphate (P i ) 1,2 is extended to include selected excitatory amino acids. The synthesis and photochemical studies of N-and ...

... P.;Rhodes,MR;McBurney,RN;Reddy, NL; Hu, L.-Y.; Cottar, RE; Hamilton, PN; Weber, E.; Keana, J ... DF; Bigge, CF; Colbry, N. L.; Johnson, G.;Leecosky, L. J.; Malone,T. C.; Michael, A.; Reily ... Density Functional Investigation of rr-Interactions, Ph.D. Thesis, University of Illinois, 1991. ...

We have developed a methodology for inducing a rapid rise in extracellular dopamine concentrations. The clearance of the applied dopamine, as well as its effect on the endogenous dopamine release (i.e., autoinhibition), was then examined using fast scan cyclic voltammetry. In a recording chamber mounted on a Nikon Optiphot epifluorescence microscope, coronal rat brain slices containing either the caudate nucleus or prefrontal cortex were perfused with ACSF containing 100-200 microM 'caged-DA.' UV illumination (100-200 ms) focused at the tip of the recording electrode produced a peak DA concentration of 1-2 microM within 100-200 ms of terminating the illumination. The caudate nucleus exhibited a faster clearance rate for photo-released DA compared to the prefrontal cortex. Cocaine reduced the clearance rates in both the caudate nucleus and prefrontal cortex. In the prefrontal cortex a combination of desipramine/clomipramine also reduced dopamine clearance, suggesting heterologous uptake of the applied DA by noradrenergic and/or serotonergic terminals. Photo-released dopamine inhibited release of endogenous caudate DA release evoked by single electrical stimulation. The advantages of this methodology are discussed.

Presynaptic calcium (Ca(pre)) has been studied extensively because of its role in triggering and modulating neurotransmitter release. Although calcium regulation and calcium-driven processes can be strongly temperature dependent, technical difficulties have limited most studies of Ca(pre) to temperatures well below the physiological range. Here we assessed the use of membrane-permeant acetoxymethyl (AM) indicators and dextran-conjugated indicators for measuring Ca(pre) at physiological temperatures. A comparison of these two types of indicators loaded into parallel fibers of rat cerebellar slices revealed striking differences. AM indicators were rapidly extruded from axons and presynaptic terminals and therefore cannot be used for long-term measurements at high temperatures. In contrast, dextran-conjugated indicators were retained within parallel fibers and are therefore well suited to measuring Ca(pre) at physiological temperatures. The limited number of dextran indicators available prompted us to synthesize three new indicators that show peak emission in the red (575-600 nm). These indicators allow for simultaneous use of multiple calcium indicators that can be readily distinguished on the basis of excitation and emission wavelengths, use of excitation and emission wavelengths that are relatively insensitive to tissue autofluorescence, and measurements in systems with expression of green fluorescent protein (GFP). Thus we find that dextran-conjugated indicators are well suited to long-term recordings of Ca(pre) at physiological temperatures and that the development of new red indicators greatly extends their utility.

IDDC (3, 10,5-(iminomethano)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene++ +) and a series of substituted derivatives were synthesized and evaluated in vitro for their ability to displace tritiated MK-801 ([3H]-2) from its specific binding site in guinea pig brain homogenate. Substitution at the 3-position of 3 with bromine, chlorine, and fluorine led to increased binding affinity. In contrast, substitution of donor groups at the 3-position gave decreased binding affinities, as did all substitutions at the 7-position and on nitrogen. Where racemic mixtures were resolved, the (+)-optical antipodes were more active than their enantiomers or racemates. The most active ligand found in this study was (+)-13e (IC50 = 15.5 +/- 4.5 nM). The affinity of (+)-13e for the PCP receptor makes it among the most potent ligands known. In vitro neuroprotection was demonstrated by 3, (+)-3, and (+)-6 (N-Me-IDDC) against glutamate-induced cell death in rat hippocampal cells.