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Biomedical subjects

A G Ewing

Publications and source records attributed to A G Ewing.

At least 37 records · Page 2Linked to original sources
PubMed1997

Ultrathin slab gel separations of DNA using a single capillary sample introduction system.

We demonstrate here a novel method for DNA separations which combines the parallel processing capabilities of slab gels with the advantages of sample introduction obtained with a single capillary. This sample introduction format allows rapid sequential separations or continuous analysis to be carried out on ultrathin slab gels with efficient heat dissipation. Ultrathin slab gels have been fabricated by using 57-micron spacers between quartz plates, and a single capillary has been used to introduce plugs of dsDNA fragments into the ultrathin gel. These fragment plugs were deposited along the entrance to the ultrathin gel at spatially discrete locations by micromanipulation of the capillary. Spatially resolved detection has been accomplished with an argon ion laser focused to a line for excitation and a CCD for collection of fluorescence. Double-stranded DNA separations are demonstrated in a plug injection format. This approach allows multiple unique samples to be rapidly deposited on the ultrathin slab gels for separation.

DNA↗
PubMed1997

Atomic and molecular imaging at the single-cell level with TOF-SIMS.

A complete cold chain freeze-fracture methodology has been developed to test the feasibility of using time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging for the molecular analysis of frozen hydrated biological samples. Because the technique only samples the first few monolayers of a sample, water on the surface of a sample can be a major source of interference. This problem can be minimized by placing a cold trap (fracture knife and housing at -196 degrees C) near the fractured sample that is held at a warmer temperature (-97 to -113 degrees C). This results in removal of surface water and prevents condensation on the surface. Although this approach is effective, it has been found that sample warming needs to be carefully controlled due to the volatility of other matrix molecules and the morphological effects imparted onto the cell surface during drying. By utilizing the above handling technique, it has been possible to demonstrate for the first time that TOF-SIMS imaging technology can be used to obtain images of molecular species across a cell surface with a submicrometer ion probe beam. Images of small hydrocarbons and the deliberately added dopants DMSO and cocaine have been obtained with TOF-SIMS of the single-cell organism Paramecium.

Animals↗
PubMed1997

Critical review of recent developments in fluorescence detection for capillary electrophoresis.

Recent developments in capillary electrophoresis with fluorescence detection are reviewed. Instrumental advances have led to increased sensitivity and, therefore, a growing number of applications. Capillary electrophoresis has been coupled with various techniques to achieve multi-dimensional separations. Other advances have focused on temporal resolution when sampling from biological environments, increased sample throughput especially for DNA analysis, and fast separation times. New technologies including chip and channel electrophoretic separations with fluorescence detection are also discussed.

DNA↗
PubMed1997

Quantitative measurements of released amines from individual exocytosis events.

Chemical analysis of single cells is an area of great interest in the biological sciences. Single-cell systems are being utilized as a model to understand in vivo processes better. One method that is moving to the forefront in cellular analysis is electrochemistry. Owing to their rapid response time and small dimensions, voltammetric microelectrode techniques, such as amperometry and fast-scan voltammetry, have made it possible to monitor minute amounts of biological compounds and transiently occurring chemical events in cellular systems. The application of these methods to the quantitation of individual vesicular release events from single cells is overviewed here. The application of electrochemical monitoring to several types of cultured cells, including bovine adrenal chromaffin cells, rat pheochromocytoma (PC12) cells, beige mouse mast cells, superior cervical ganglion neurons, and human pancreatic beta-cells, as well as to the invertebrate systems, the leech Hirudo medicinalis, and pond snail Planorbis corneus has provided a wealth of new information concerning exocytosis. Results obtained from the studies highlight the potential of electrochemical techniques in cellular analysis to contribute to our understanding of molecular and pharmacological effects on exocytosis. This article overviews work done on all the above cell types with an emphasis on PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1997

Chemical analysis of single cells and exocytosis.

Recent advances in the development of microscale analytical techniques have allowed minute amounts of compounds of biological interest to be monitored in microenvironments. Microcolumn separation techniques, such as microcolumn liquid chromatography and capillary electrophoresis, provide a means of obtaining high-efficiency separations of analytes in picoliter to femtoliter volume samples. Voltammetric microelectrode techniques, such as amperometry and fast-scan cyclic voltammetry, are ideally suited for monitoring transiently occurring chemical events in cellular and subcellular processes owing to their rapid response times and small structural dimensions. The principles and applications of these techniques in single-cell analysis are discussed throughout this review. Multicomponent separations and quantitations of large invertebrate neurons of the land snail Helix neurons and the pond snail Planorbis corneus dopamine neurons, and of small mammalian cells, bovine adrenal cells, rat PC12 cells, and human lymphocytes, by use of microcolumn liquid chromatography and capillary electrophoresis are presented. Electrochemical monitoring of neurotransmitter exocytosis from single adrenal cells, from PC12 cells, and from the cell body of the Planorbis dopamine neuron is highlighted. Results obtained from both separation and voltammetric techniques in single-cell analyses will provide a better understanding of cellular and/or neuronal chemistry and biology in complicated living environments.

Adrenal Medulla↗
PubMed1996

Flow injection analysis using continuous channel electrophoresis.

Continuous zone electrophoretic separations in narrow channels coupled to small-bore capillaries have been demonstrated and characterized previously. Presented here is the use of this new technique to monitor dynamic chemical changes occurring in a flow injection analysis system. The fundamental aspects of the data that this type of separation generates are discussed in a comparison of static and dynamic analyses. An analysis of a dynamic separation is also provided to thoroughly outline the steps necessary to deconvolute the data. Three types of dynamic analyses, which simulate realistic analytical situations, are then examined in detail. The first of these involves the addition of a mixture of four dansylated amino acids to the flow injection system to provide several different sample duration periods. The second is the staggered addition of short-duration plugs of analyte to the system. The third is the continuous addition of one analyte at different concentrations. Quantitative information generated by these experiments includes the simultaneous determination of the time of analyte contact and its duration of contact with the sampling capillary, identification of analytes based on electrophoretic mobilities, and concentration changes with time. For the addition of a 1.52 mM solution of N epsilon-dansyl-L-lysine, the sample duration and time of analyte contact with the capillary was determined with an error of < 4%. Concentration changes in the FIA system of dansyl-L-arginine over the range of 0.38-3.04 mM are also demonstrated with transitional edges on the time scale of 3-4 s. Qualitatively, the appearance of analyte bands can reveal impurities and fronting or tailing effects as seen in conventional capillary electrophoretic separations. The results demonstrate the ability of the technique to successfully probe dynamic environments.

Arginine↗
PubMed1996

Electrochemical monitoring of bursting exocytotic events from the giant dopamine neuron of Planorbis corneus.

We have discovered a neuronal system that fires bursting exocytotic events. In the giant dopamine neuron of the fresh water snail Planorbis corneus, bursting exocytotic events are evoked following in situ stimulation with elevated potassium. Amperometric detection using carbon fiber microelectrodes, which provides high temporal resolution, has been used to record exocytotic events released from the neuron. Evaluation of the time interval between consecutive exocytotic events (inter-spike interval) recorded from about 80% of the neurons reveals the occurrence of distinct bursting patterns defined by transients having an equal interval among them. Statistical analysis of these bursting exocytotic events shows three distinct distributions of inter-spike intervals with mid points occurring at 5, 22 and 45 ms. This bursting release behavior is not observed from cultured pheochromocytoma cells although they show calcium-dependent exocytosis following in situ stimulation with elevated potassium. Our data appear to indicate that the Planorbis dopamine neuron in vivo is actively involved in specific modes of neural communication and may represent an important phenomenon in understanding single cell activities.

Action Potentials↗
PubMed1996

Electrochemical monitoring of individual exocytotic events from the varicosities of differentiated PC12 cells.

Rat pheochromocytoma (PC12) cells have been used as a model of developing neurons to study exocytosis during differentiation. Upon treatment with nerve growth factor, PC12 cells become more neuronal-like. Using amperometric detection at carbon fiber microelectrodes, time-resolved exocytosis of electroactive catecholamines can be observed. The site of exocytosis has been compared for differentiated and undifferentiated cells. Upon differentiation, cells release catecholamines primarily from varicosities along their neurites with no release from the cell body. In addition, the mean vesicular content is not significantly altered upon differentiation although it appears that the distribution of vesicle content becomes more narrow. The number of release events observed also decreases as the cells become more neuronal in character. It is possible that the smaller range of vesicle dopamine content and the decreased number of release events observed after differentiation are a result of the relocation of the site of exocytosis.

Animals↗
PubMed1996

Analysis of single cells with capillary electrophoresis electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Recent results with on-line capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry suggest that CE/ESI-FTICR can provide a powerful technique for micro-sample analyses owing to the inherent sensitivity of the technique and the enhanced information content derived from high-performance mass measurements. Using micro-sampling methods and ion accumulation techniques based on quadrupolar excitation, we demonstrate that adequate sensitivity exists to characterize the hemoglobin from a single human erythrocyte (approximately 450 amol). In these studies mass spectra with average mass resolution in excess of 45 000 (FWHM) were obtained for both the alpha- and beta chain of hemoglobin following in-column lysing of a single erythrocyte.

Cyclotrons↗
PubMed1996

The latency of exocytosis varies with the mechanism of stimulated release in PC12 cells.

To compare the time course of different mechanisms of chemically stimulated release, amperometric detection of dopamine was carried out at single PC12 cells. The rapid response of carbon fiber microelectrodes allowed the detection of single exocytotic events, thus providing time-resolved information about the dynamics of stimulated release, in particular the latency between the stimulation of a cell and the secretion of catecholamines. On rapid depolarization of the cell membrane caused by application of 105 mM K+, almost immediate (6 +/- 1 s) release of dopamine was observed. Stimulation with 1 mM nicotine, involving the stimulant binding to a ligand-gated ion channel, resulted in a short (37 +/- 5 s) delay between stimulation and secretion. Application of 1 mM muscarine to the cells caused a long (103 +/- 11 s) latency before exocytosis was detected. A biphasic response that appeared to be similar to a combination of nicotine- and muscarine-stimulated release was observed when cells were stimulated with 10 mM acetylcholine. Thus, it appears that the dynamics of stimulated release at single PC12 cells is significantly affected by the mechanism leading to exocytosis.

Acetylcholine↗
PubMed1996

Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic beta-cells.

Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic beta-cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for < 6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine-containing vesicles at later days in culture is responsible for this alteration.

Animals↗
PubMed1995

Multiple classes of catecholamine vesicles observed during exocytosis from the Planorbis cell body.

The heterogeneous nature of vesicles has been studied by evaluating exocytotic events released from the cell body of a dopamine-containing neuron of Planorbis corneus. Vesicular exocytosis has been elicited by stimulation with in situ application of elevated potassium and has been monitored electrochemically with a carbon fiber microelectrode placed on the cell body. These electrodes allow individual release events to be monitored and quantitated to reveal vesicular dopamine content. Statistical analysis of individual release events demonstrates that two classes of vesicles with specific bimodal distributions in dopamine content and vesicle size are observed after cell stimulation. The effect of a psychostimulant on individual vesicular dopamine level has been studied by treating the cells with D-amphetamine. After a 20-min application of 10 microM amphetamine, changes in both vesicle content and size distributions are obtained with an overall decrease in vesicular dopamine level of 40%. The altered distribution show trimodal shapes indicating a third class of vesicles is created by amphetamine treatment. Our data appears to indicate that multiple classes of vesicles are released from the cell body of the dopamine-containing neuron and vesicular dopamine level can be manipulated by the application of the lipophilic weak base amphetamine.

Amphetamine↗
PubMed1995

Capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for direct analysis of cellular proteins.

The combination of capillary electrophoresis (CE) with electrospray ionization (ESI) mass spectrometry has proven to be broadly applicable to a wide range of biologically important compounds. When combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, the combined method, in addition to high-resolution separations, affords high-resolution precision mass measurements for analytes separated from complex mixtures. Direct chemical analysis of single cells has received considerable attention in recent years; the single cell approach provides a major step toward answering important questions in the field of cellular biochemistry. In this work we present preliminary results which demonstrate the feasibility of using the CE-ESI-FTICR combination as a high-performance detection scheme for the analysis of cellular proteins acquired directly from small populations (i.e., 5-10) of intact living cells. The human erythrocyte was chosen as a model system owing to its availability, relatively homogeneous composition, and thorough documentation of contents by previous researchers. In this work we demonstrate the on-line acquisition of high-resolution mass spectra (average resolution > or = 45,000 fwhm) of both the alpha and the beta chains of hemoglobin acquired from the injection of 10 human erythrocytes (corresponding to 4.5 fmol of hemoglobin). Given the extremely small volume of the human erythrocyte (typically 87 fl/cell), the techniques implemented here should also be adaptable to the study of larger mammalian cell systems.

Adult↗
PubMed1995

Analysis of single cells by capillary electrophoresis with on-column derivatization and laser-induced fluorescence detection.

On-column derivatization of single mammalian cells with capillary electrophoretic separation and laser-induced fluorescence detection is described. Individual cells are electrophorectically injected into the front end of the separation capillary, which is used as a chamber to lyse the cell and derivatize its contents for subsequent separation and detection. Reagents to lyse the cell and derivatize its contents are electrophoretically injected into the front end of the capillary (2.7 mm, 600-pL volume for a 17-microns-i.d. capillary) after the cell has been injected. Dopamine and five amino acids have been quantitatively determined in individual rat pheochromocytoma cells after on-column derivatization with naphthalene-2,3-dicarboxaldehyde and CN-. Average values of compounds determined in these cells range from 180 +/- 110 amol/cell for aspartic acid to 5.1 +/- 1.5 fmol/cell for taurine.

Amines↗
PubMed1995

Observation and quantitation of exocytosis from the cell body of a fully developed neuron in Planorbis corneus.

We have discovered a neuronal system that releases neurotransmitter via exocytosis from the cell body. In the large dopamine cell of the pond snail Planorbis corneus, depolarization induces rhythmic release of dopamine from the cell body. When a stimulant is applied extracellularly or intracellularly in situ to the cell body, transient dopamine concentration packets that appear in a bursting pattern are observed. Dopamine release is calcium dependent and release is on the time scale expected for exocytosis (2 to 4 msec rise times). Quantitation of individual events reveals an average of 818,000 molecules per exocytotic event. As many as 89,000 individual exocytotic events have been observed following a single stimulation of one cell. Neurotransmitter exocytosis from the neuronal cell body appears to represent an alternative form of neurocommunication to synaptic transmission.

Animals↗
PubMed1994

Analysis of human cerebrospinal fluid by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with laser-induced fluorescence detection is used to analyze 10 microL samples of human cerebrospinal fluid. Primary amine-containing compounds in untreated cerebrospinal fluid are labeled with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde prior to analysis, producing fluorescent isoindoles. Electropherograms containing approximately 50 peaks are obtained in less than 35 min from cerebrospinal fluid samples. Ten peaks in the electropherograms have been identified and quantitated: arginine, glutamine, threonine, valine, gamma-amino-n-butyric acid, serine, alanine, glycine, glutamic acid, and aspartic acid. Detection limits for these 10 amino acids range from 0.29 nM for gamma-amino-n-butyric acid to 100 nM for threonine, and separation efficiencies as high as 190,000 theoretical plates are obtained for these analytes. Electropherograms of cerebrospinal fluid samples from patients with Alzheimer's disease and from children with different neurological disorders are compared to those of healthy controls. Differences in individual amino acid levels are observed between the patient groups, and these differences appear to be disease and age related. These results indicate that analysis of cerebrospinal fluid by capillary electrophoresis will be useful as a selective, rapid, and sensitive tool for both diagnosis of central nervous system disorders and for study of the mechanisms of these disorders.

Adult↗
PubMed1994

Amperometric monitoring of stimulated catecholamine release from rat pheochromocytoma (PC12) cells at the zeptomole level.

Catecholamine release from rat pheochromocytoma (PC12) cells has been observed at zeptomole levels using dc-amperometric detection with carbon fiber microelectrodes. Time-resolved individual exocytic events from PC12 cells have been recorded and analyzed with 1.2 ms time resolution. The average area under 1912 current transients from 13 PC12 cells corresponds to 190 zmol (114,300 molecules per release event). The average width at half-height of these current transients is 9.3 ms, in agreement with the time frame of exocytosis. The detection limit of the method reported here is as low as 31 zmol. This is the first report of direct electrochemical observation of quantal release from PC12 cells. The successful application of this electrochemical scheme to monitor catecholamines released from small vesicles also suggests that it may be possible to apply this technique to monitor quantal release from synaptic vesicles.

Animals↗