Search PubMed⌕ Search

Biomedical subjects

R T Kennedy

Publications and source records attributed to R T Kennedy.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detection of multiple patterns of oscillatory oxygen consumption in single mouse islets of Langerhans.

A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 +/- 1.17, 9.21 +/- 2.15, and 12.22 +/- 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean +/- SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 +/- 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 +/- 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca2+-channel blocker nifedipine (40 microM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca2+. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca2+ levels and are suggestive of an important role for Ca2+ in amplifying metabolic oscillations.

Animals↗
PubMed1999

Insulin-stimulated insulin secretion in single pancreatic beta cells.

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.

Animals↗
PubMed1999

Detection of peptides by precolumn derivatization with biuret reagent and preconcentration on capillary liquid chromatography columns with electrochemical detection.

The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25-micron inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-microL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus.

Animals↗
PubMed1999

Identification, quantitation, and characterization of biomolecules by capillary electrophoretic analysis of binding interactions.

The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis.

Binding, Competitive↗
PubMed1999

Quantitative analysis of receptors for adenosine nucleotides obtained via in vitro selection from a library incorporating a cationic nucleotide analog.

5-(3"-Aminopropynyl)-2'-deoxyuridine (dJ), a modified nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.

Adenine Nucleotides↗
PubMed1998

Aptamers as ligands in affinity probe capillary electrophoresis.

A DNA aptamer against IgE was labeled with fluorophore and used as a selective fluorescent tag for determining IgE by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). CE-LIF separations of samples containing fluorescently labeled aptamer and IgE were complete in less than 60 s and revealed two zones, one corresponding to free aptamer and the other to aptamer bound to IgE. The free aptamer peak decreased and bound aptamer peak increased in proportion to the amount of IgE in the sample so that IgE could be detected with a linear dynamic range of 10(5) and a detection limit of 46 pM. The assay was highly selective as aptamer was unaffected by the presence of IgG and IgE did not bind other DNA sequences. IgE was determined in serum samples with similar analytical figures of merit. Similar conditions using a thrombin aptamer allowed detection of thrombin.

Affinity Labels↗
PubMed1998

On-line competitive immunoassay based on capillary electrophoresis applied to monitoring insulin secretion from single islets of Langerhans.

An automated on-line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate-labeled insulin (FITC-insulin), monoclonal anti-insulin and perifusate of single islets were mixed on-line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC-insulin for antibody binding sites. Therefore, the amounts of bound and free FITC-insulin were modulated by insulin released from islets. The bound and the free FITC-insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme-linked immunoassay. This on-line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody-antigen reaction rate and binding equilibrium were also studied.

Animals↗
PubMed1998

Determination of trace level gamma-aminobutyric acid using an improved OPA pre-column derivatization and on-column preconcentration capillary liquid chromatography with electrochemical detection.

An improved pre-column derivatization with o-phthalaldehyde/tert-butylthiol and on-column preconcentration are used with packed capillary liquid chromatography and electrochemical detection to obtain low concentration detection limits for gamma-aminobutyric acid (GABA). Using derivatization procedures from the literature, it was found that the detection limits for GABA were 380 amol in 50 microns i.d. packed capillaries, which is over 10-fold worse than the detection limit possible with the instrumentation. The higher detection limit was directly the result of electroactive interferences generated by the derivatization chemistry. Derivatization was improved by scavenging excess reagents with excess amine and iodoacetamide. With these improvements, the interfering peaks were eliminated and the detection limit was improved to 50 amol. With injection volumes of 0.5 microL, under conditions that permitted on-column preconcentration, the concentration detection limit, that is the concentration at which analytes could be derivatized and detected, was 100 pM. The technique was applied to determination of GABA release from islets of Langerhans.

Animals↗
PubMed1998

Evidence for neuronal origin and metabotropic receptor-mediated regulation of extracellular glutamate and aspartate in rat striatum in vivo following electrical stimulation of the prefrontal cortex.

Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5-s intervals in the striatum of chloral hydrate-anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser-induced fluorescence. Application of a single 10-s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200-300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 microM tetrodotoxin or depletion of extracellular Ca2+, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (200 microM) increased resting Glu and Asp levels by 300-500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylate (200 microM). This effect was blocked by the metabotropic Glu receptor antagonist (RS)-alpha-methylcarboxyphenylglycine (MCPG; 200 microM). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of L-trans-pyrrolidine-2,4-dicarboxylic acid, MCPG produced a five- to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.

Animals↗
PubMed1997

Monitoring of met-enkephalin in vivo with 5-min temporal resolution using microdialysis sampling and capillary liquid chromatography with electrochemical detection.

A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 microl/min. Separations were performed using 7 cm x 25 microm I.D. fused-silica capillary columns packed with 5 microm Alltima C18 particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 microl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K+-induced depolarization conditions.

Animals↗
PubMed1997

Effects of intravesicular H+ and extracellular H+ and Zn2+ on insulin secretion in pancreatic beta cells.

The effects of extracellular Zn2+ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H+-ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn2+ concentration from 0 to 25 microM increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn2+ effects were likely exerted through a common-ion effect on Zn2+-insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.

Animals↗
PubMed1997

High temporal resolution monitoring of glutamate and aspartate in vivo using microdialysis on-line with capillary electrophoresis with laser-induced fluorescence detection.

An automated method for high temporal resolution monitoring of the neurotransmitters glutamate and aspartate in vivo using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was developed. Microdialysis probes placed in the striatum of anesthetized rats were coupled on-line with the CE system by an automated flow-gated interface. Analytes were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and detected by LIF using the 354 nm line (7 mW) of a He-Cd laser for excitation. With dialysis flow rates of 1.2 microL/ min, the detection limit at the dialysis probe was 200 nM for glutamate and aspartate. Glutamate and aspartate could be resolved in less than 5 s with over 200,000 theoretical plates. The sampling time was limited by the separation time while the temporal resolution was limited to approximately 12 s because of band broadening that occurs within the probe and its associated tubing. The high temporal resolution allowed the first simultaneous monitoring of glutamate and aspartate overflow during acute electrical stimulation in the rat brain.

Animals↗
PubMed1997

Insulin-like growth factor II signaling through the insulin-like growth factor II/mannose-6-phosphate receptor promotes exocytosis in insulin-secreting cells.

The insulin-like growth factor II (IGF-II)/mannose-6-phosphate (M-6-P) receptor is known to participate in endocytosis as well as sorting of lysosomal enzymes and is involved in membrane trafficking through rapid cycling between cytosolic membrane compartments and the plasma membrane. Here we demonstrate that IGF-II, acting through the IGF-II/M-6-P receptor, promotes exocytosis of insulin in the pancreatic beta cell. The effect of IGF-II was evoked at nonstimulatory concentrations of glucose, was mediated by a pertussis toxin sensitive GTP-binding protein, was dependent on protein kinase C-induced phosphorylation, and was independent of changes in cytoplasmic free Ca2+ concentration. Since the applied concentration of IGF-II is within the range normally found free in circulation in humans, this novel signaling pathway for the IGF-II/M-6-P receptor is likely to be involved in modulation of insulin exocytosis under physiological conditions.

Adenosine Triphosphate↗
PubMed1997

In vivo monitoring of glutathione and cysteine in rat caudate nucleus using microdialysis on-line with capillary zone electrophoresis-laser induced fluorescence detection.

A fully-automated method for monitoring thiols in vivo using microdialysis coupled on-line with capillary zone electrophoresis with laser-induced fluorescence detection was developed. Dialysates were derivatized on-line with monobromobimane and automatically transferred to the separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nl/min resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits for these analytes were in the 20-40 nM range and the response was linear up to 20 microM. The system was applied to the measurement of glutathione and cysteine in the extracellular space of the caudate nucleus of anesthetized rats. The measured basal concentrations of glutathione and cysteine were 2.0 +/- 0.1 microM and 2.3 +/- 0.3 microM, respectively which agree well with literature values. Increases in glutathione and cysteine were monitored with 180 s temporal resolution during stimulation by infusion of potassium. The average concentration of glutathione and cysteine during stimulation was 3.0 +/- 0.9 and 3.3 +/- 0.5 microM (n = 3), respectively. This system is the first to obtain high relative recoveries and high temporal resolution simultaneously for multiple thiols with microdialysis sampling in the brain.

Animals↗
PubMed1997

Dual microcolumn immunoassay applied to determination of insulin secretion from single islets of Langerhans and insulin in serum.

A dual microcolumn immunoassay (DMIA) was developed and applied to determination of insulin in biological samples. The DMIA utilized a protein G capillary column (150 microns I.D.) with covalently attached anti-insulin to selectively capture and concentrate insulins in a sample. Insulins retained in the capillary immunoaffinity column were desorbed and injected onto a reversed-phase capillary column (150 microns I.D.) for further separation from interferences such as cross-reactive antigens and non-specifically adsorbed sample components. Bovine, porcine and rat insulin all cross-reacted with the antibody and could be determined simultaneously. Using a UV absorbance detector, the dual microcolumn system had a detection limit of 10 fmol or 20 pM for 500-microliter sample volumes. The DMIA system was used to measure glucose-stimulated insulin secretion from single rat islets of Langerhans. Because of the separation in the second dimension, both rat I and rat II insulin could be independently determined. The system was also evaluated for determination of insulin in serum. Using microcolumns instead of conventional HPLC columns resulted in several advantages including use of less chromatographic material and improved mass detection limit.

Animals↗
PubMed1997

Measurement of antibody-antigen dissociation constants using fast capillary electrophoresis with laser-induced fluorescence detection.

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate (FITC)-labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Kd determinations were made by separating free FITC-insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC-insulin in the picomolar range, as is required to measure Kd's below 1 nM. The Kd of FITC-insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The Kd's of the antibody with unlabeled insulins from several species were obtained by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC-insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. Kd's for the different insulins were between 0.34 and 0.64 nM.

Animals↗
PubMed1996

On-line competitive immunoassay for insulin based on capillary electrophoresis with laser-induced fluorescence detection.

An on-line competitive immunoassay for insulin has been developed and applied to monitoring insulin concentration in a flowing stream. In the assay, solutions of fluorescein-labeled insulin (FITC-insulin), monoclonal anti-insulin, and sample containing insulin are pumped into a cross where they begin to mix. The mixture flows through a fused silica reactor capillary to a flow-gated interface. During transfer to the interface, insulin and FITC-insulin compete to form a complex with the antibody. At the interface, a plug of the mixture is injected into a separation capillary, where the bound and free FITC-insulin are separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The amount of bound FITC-insulin, amount of free FITC-insulin, or bound/free ratio can be used to quantify insulin concentration. Typical relative standard deviations of bound over free ratio are 5%. The detection limit of the immunoassay in the on-line mode is < 0.3 nM. Each separation requires as little as 3 s, and over 1600 consecutive assays can be acquired with no need to rinse the separation capillary. Thus, the system can be used to monitor insulin in a flowing stream for flow injection analysis or for sensor-like monitoring. Dilution and zone broadening during transfer of sample to the interface limit the response time of the on-line system to about 25 s. As a demonstration of the on-line immunoassay, the insulin content of single islets of Langerhans was determined by flow injection analysis.

Electrophoresis, Capillary↗