Craig A Aspinwall
PMID: 23499741;PMCID: PMC3628080;Abstract:
Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca2+ concentration ([Ca2+]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca2+-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca2+ influx since exclusion of extracellular Ca2+ diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape. © 2013 Elsevier Inc.
Black lipid membranes (BLMs) are widely used for recording the activity of incorporated ion channel proteins. However, BLMs are inherently unstable structures that typically rupture within a few hours after formation. Here, stabilized BLMs were formed using the polymerizable lipid bis-dienoyl phosphatidylcholine (bis-DenPC) on glass pipettes of approximately 10 microm (I.D.). After polymerization, these BLMs maintained steady conductance values for several weeks, as compared to a few hours for unpolymerized membranes. The activity of an ion channel, alpha-hemolysin, incorporated into bis-DenPC BLMs prior to polymerization, was maintained for 1 week after BLM formation and polymerization. These lifetimes are a substantial improvement over those achievable with conventional BLM technologies. Polymerized BLMs containing functional ion channels may represent an enabling technology for development of robust biosensors and drug screening devices.
PMID: 22911376;PMCID: PMC3716455;Abstract:
Photolytic optical gating (POG) facilitates rapid, on-line and highly sensitive analyses, though POG utilizes UV lasers for sample injection. We present a low-cost, more portable alternative, employing an ultraviolet light-emitting diode (UV-LED) array to inject caged fluorescent dyes via photolysis. Utilizing the UV-LED array, labeled amino acids were injected with nanomolar limits of detection (270 ± 30 nM and 250 ± 30 nM for arginine and citrulline, respectively). When normalized for the difference in light intensity, the UV-LED array provides comparable sensitivity to POG utilizing UV lasers. Additionally, the UV-LED array yielded sufficient beam quality and stability to facilitate coupling with a Hadamard transform, resulting in increased sensitivity. This work shows, for the first time, the use of an UV-LED for online POG with comparable sensitivity to conventional laser sources but at a lower cost. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and -0.85V vs. SSCE at 100 V s-1 in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 μM (five points) had slopes of 72 ± 2 pA μM-1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23 nM and response time (t90%) was 40ms or less. The electrode response to 0.2 μM insulin was stable for 3 days. The modified electrode was used for amperometric detection of exocytosis from individual pancreatic β-cells.
Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH2) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH2 after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH2 with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH2 was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH2 was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH2 in cells treated with an HNO donor suggests that the concentration of GS(O)NH2 can be correlated to intracellular levels of HNO.