A photoluminescence-based field method for detection of traces of explosives.
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We report a photoluminescence-based field method for detecting traces of explosives. In its standard version, the method utilizes a commercially available color spot test kit for treating explosive traces on filter paper after swabbing. The colored products are fluorescent under illumination with a laser that operates on three C-size flashlight batteries and delivers light at 532 nm. In the fluorescence detection mode, by visual inspection, the typical sensitivity gain is a factor of 100. The method is applicable to a wide variety of explosives. In its time-resolved version, intended for in situ work, explosives are tagged with europium complexes. Instrumentation-wise, the time-resolved detection, again visual, can be accomplished in facile fashion. The europium luminescence excitation utilizes a laser operating at 355 nm. We demonstrate the feasibility of CdSe quantum dot sensitization of europium luminescence for time-resolved purposes. This would allow the use of the above 532 nm laser. (+info)
The effect of body condition on disposition of alkaloids from silvery lupine (Lupinus argenteus pursh) in sheep.
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Several species of lupine (Lupinus spp.) are poisonous to livestock, producing death in sheep and "crooked calf disease" in cattle. Range livestock cope with poisonous plants through learned foraging strategies or mechanisms affecting toxicant disposition. When a toxic plant is eaten, toxicant clearance may be influenced by the animal's nutritional and/or physiological status. This research was conducted to determine whether differences in body condition or short-term nutritional supplementation of sheep altered the disposition of lupine alkaloids given as a single oral dose of ground silvery lupine (Lupinus argenteus) seed. Ewes in average body condition (ABC, n = 9) and low body condition (LBC, n = 10) received a single dose of ground lupine seeds including pods (8.5 g/kg BW) via gavage on the first day of the experiment, and were then randomly assigned to one of two nutritional supplement treatments. Blood samples were taken 0 to 60 h after dosing to compare blood alkaloid concentration and to evaluate alkaloid absorption and elimination profiles. Concentrations of total alkaloid and anagyrine, 5,6 dehydrolupanine, lupanine, and alkaloid E were measured in serum. These four alkaloids constituted 78 and 75% of the total alkaloid concentration in serum for LBC vs. ABC groups, respectively. Initial analysis indicated that short-term supplementation had no effect on alkaloid disposition, and supplementation was removed from the statistical model. The highest concentration of total alkaloids was observed 2 h after dosing. Overall, serum total alkaloid and anagyrine levels (area under the curve) were higher (P < 0.01) for sheep in the LBC group. Serum peak concentrations of total alkaloid and anagyrine were higher in LBC vs. ABC groups (P < 0.05). Serum elimination of anagyrine, unknown alkaloid E, and lupanine was decreased in LBC vs. ABC treatments (P < 0.05). These results demonstrate that body condition is important in the disposition of lupine alkaloids; however, further research is needed to determine the potential benefit, if any, that short-term nutritional supplementation might have on alkaloid disposition. (+info)
Subunit composition of nicotinic receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA.
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Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, alpha2, alpha4, alpha6, beta2, and beta3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of alpha3 nAChR subunit-immunoreactivity in this same region, but not that for alpha5 and beta4. Relatively high levels of alpha2 and alpha3 subunits were also identified in monkey cortex, in addition to alpha4 and beta2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased alpha6 and beta3 subunit immunoreactivity by approximately 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, alpha3, alpha4, and beta2 subunit immunoreactivity was decreased approximately 50%, whereas alpha2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([(3)H]cytisine, (125)I-alpha-bungarotoxin, and (125)I-alpha-conotoxin MII) suggest the following: that alpha6beta2beta3, alpha6alpha4beta2beta3, and alpha3beta2* nAChR subtypes are present on dopaminergic terminals and that the alpha4beta2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas alpha2beta2* and alpha7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-alpha7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease. (+info)
Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization.
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The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with a4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering a4* receptors hypersensitive to nicotine. Selective activation of a4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of a4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization. (+info)
A robust homogeneous binding assay for alpha4beta2 nicotinic acetylcholine receptor.
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AIM: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators. METHODS: Membrane preparation of HEK293 cells expressing alpha4beta2 nAChR, [(3)H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. RESULTS: IC(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [(3)H]cytisine binding to alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. CONCLUSIONS: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels. (+info)
Suppression of laser-induced choroidal neovascularization by oral administration of SA3443 in mice.
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The effect of a synthetic cyclic disulfide compound, SA3443, on neovascularization was investigated. In vitro, enzyme-linked immunosorbent assay and RT-PCR demonstrated that SA3443 suppressed the expression of the hypoxia-induced vascular endothelial growth factor (VEGF) at both protein and mRNA levels in ARPE-19 cells. In vivo, the administration of SA3443 to mice with laser-induced choroidal neovascularization (CNV) suppressed the leakage from the lesions and reduced their size. Furthermore, the expression level of VEGF protein was significantly reduced by the administration of SA3443. Taken together, our results demonstrate that SA3443 suppresses VEGF production and reduces vascular leakage and the growth of mouse experimental CNV. (+info)
Long-term exposure to the new nicotinic antagonist 1,2-bisN-cytisinylethane upregulates nicotinic receptor subtypes of SH-SY5Y human neuroblastoma cells.
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Nicotinic drug treatment can affect the expression of neuronal nicotinic acetylcholine receptors (nAChR) both in vivo and in vitro through molecular mechanisms not fully understood. The present study investigated the effect of the novel cytisine dimer 1,2-bisN-cytisinylethane (CC4) on nAChR natively expressed by SH-SY5Y neuroblastoma cells in culture. CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC50=220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3H-epibatidine (3H-Epi; 3-4-fold) or 125I-alpha-bungarotoxin (125I-alphaBgtx; 1.2-fold) sensitive receptors present on the cell membrane and in the intracellular pool. Comparable data were obtained with nicotine or cytisine, but not with carbamylcholine, d-tubocurarine, di-hydro-beta-erythroidine or hexametonium. Immunoprecipitation and immunopurification studies showed that the increase in 3H-Epi-binding receptors was due to the enhanced expression of alpha3beta2 and alpha3beta2beta4 subtypes without changes in subunit mRNA transcription or receptor half-life. The upregulation was not dependent on agonist/antagonist properties of the drugs, and did not concern muscarinic or serotonin receptors. Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers alpha-conotoxin MII or methyllycaconitine. In conclusion, chronic treatment with CC4 increased the number of nAChRs containing beta2 and alpha7 subunits on the plasma membrane, where they were functionally active. In the case of beta2-containing receptors, we propose that CC4, by binding to intracellular receptors, triggered a conformational reorganisation of intracellular subunits that stimulated preferential assembly and membrane-directed trafficking of beta2-containing receptor subtypes.. (+info)
Mouse beta-TC6 insulinoma cells: high expression of functional alpha3beta4 nicotinic receptors mediating membrane potential, intracellular calcium, and insulin release.
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Nicotine elicited membrane depolarization, elevation of intracellular calcium, rubidium efflux, and release of insulin from mouse beta-TC6 insulinoma cells. Such responses were blocked by the nicotinic antagonist mecamylamine but not by the muscarinic antagonist atropine. Neither the selective alpha4beta2 antagonist dihydro-beta-erythroidine nor the selective alpha7 antagonist methyllycaconitine significantly blocked the nicotine-elicited depolarization or the calcium response. The elevation of intracellular calcium did not occur in calcium-free media, indicating that the increase in intracellular calcium was due to the influx of calcium. The rank order of potency for nicotinic agonists was as follows: epibatidine > nicotine = 3-(azetidinylmethoxy)pyridine (A-85380), cytisine, dimethylphenylpiperazinium (DMPP). Cytisine and DMPP seemed to be partial agonists. The density of nicotinic receptors measured by [3H]epibatidine binding was 7-fold higher in membranes from beta-TC6 cells than in rat brain membranes. No binding of 125I-A-85380 was detected, indicating the absence of beta2-containing receptors. Reverse transcription-polymerase chain reaction analyses indicated the presence of mRNA for alpha3 and alpha4 subunits and beta2 and beta4 subunits in beta-TC6 cells. The binding and functional data suggest that the major nicotinic receptor is composed of alpha3 and beta4 subunits. The beta-TC6 cells thus provide a model system for pharmacological study of such nicotinic receptors. (+info)