Use of micellar electrokinetic chromatography to measure palmitoylation of a peptide.

Palmitoylation is the thioester linkage of the fatty acid, palmitate (C16:0), to cysteine residues on a protein or peptide. This dynamic and reversible post-translational modification increases the hydrophobicity of proteins/peptides, facilitating protein-membrane interactions, protein-protein interactions and intracellular trafficking of proteins. Manipulation of palmitoylation provides a new mechanism for control over protein location and function, which may lead to better understanding of cell signaling disorders, such as cancer. Unfortunately, few methods exist to quantitatively monitor protein or peptide palmitoylation. In this study, a capillary electrophoresis-based assay was developed, using MEKC, to measure palmitoylation of a fluorescently-labeled peptide in vitro. A fluorescently-labeled peptide derived from the growth-associated protein, GAP-43, was palmitoylated in vitro using palmitoyl coenzyme A. Formation of a doubly palmitoylated GAP-peptide product was confirmed by mass spectrometry. The GAP-peptide substrate was separated from the palmitoylated peptide product in less than 7 min by MEKC. The rate of in vitro palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development.

Impact of microdialysis probes on vasculature and dopamine in the rat striatum: a combined fluorescence and voltammetric study.

Measuring extracellular dopamine in the brain of living animals by means of microdialysis and/or voltammetry is a route towards understanding both normal brain function and pathology. Previous reports, however, suggest that the tissue response to implantation of devices may affect the outcome of the measurements. To address the source of the tissue response and its impact on striatal dopamine systems microdialysis probes were placed in the striatum of anesthetized rats. Images obtained by dual-label fluorescence microscopy show signs of ischemia and opening of the blood-brain barrier near the probe tracks. Opening of the blood-brain barrier was further examined by determining dialysate concentrations of carbi-DOPA, a drug that normally does not penetrate the brain. Although carbi-DOPA was recovered in brain dialysate, it did not alter dialysate dopamine levels or evoked dopamine release as measured by voltammetry near the probes. Microdialysis probes also significantly diminished the effect of intrastriatal infusion of kynurenate on extracellular dopamine levels as measured by voltammetry near the probes.

Chemical analysis of single cells.

Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

Theory for the impact of basal turnover on dopamine clearance kinetics in the rat striatum after medial forebrain bundle stimulation and pressure ejection.

Although microdialysis measurements suggest that extracellular dopamine concentrations in the rat striatum are in the low nanomolar range, some recent voltammetry studies suggest that the concentration may be considerably higher, perhaps in the micromolar range. The presence of such high dopamine levels in the extracellular space has to be rationalized with the rapid, linear clearance of extracellular dopamine observed after electrical stimulation of the medial forebrain bundle. Kinetic analysis of dopamine clearance after evoked release suggests that the basal extracellular dopamine concentration is below the K(M) of dopamine uptake, which is near 0.2 microm. However, dopamine clearance after pressure ejection of dopamine into the rat striatum is slow and non-linear, which may alternatively be a sign that basal dopamine release is only slightly slower than the maximal velocity of dopamine uptake, Vmax. A high basal extracellular dopamine concentration would exist if basal dopamine release were only slightly slower than the Vmax of uptake. This report introduces a new kinetic analysis of dopamine uptake that sheds light on the possible source of the different clearance rates observed following evoked dopamine release and dopamine pressure ejection. Furthermore, the analysis rationalizes the rapid dopamine clearance after evoked release with the possibility that basal extracellular dopamine levels are above the K(M) of the transporter.

Voltammetric study of extracellular dopamine near microdialysis probes acutely implanted in the striatum of the anesthetized rat.

Establishing in vivo microdialysis methods for the quantitative determination of dopamine concentrations in the extracellular space of the brain is an important yet challenging objective. The source of the challenge is the difficulty in directly measuring the microdialysis recovery of dopamine during an in vivo experiment. The recovery value is needed for quantitative microdialysis, regardless of whether conventional or no-net-flux methods are used. Numerical models of microdialysis that incorporate both diffusion and active transport processes suggest that dopamine recovery is strongly affected by processes occurring in the tissue closest to the probe. Some evidence suggests that the tissue adjacent to the probe becomes disrupted during probe implantation. Hence, the objective of the present study was to further identify whether the tissue adjacent to the probe is disrupted and, if so, whether that disruption might affect dopamine recovery. The experiments were conducted with microdialysis probes implanted acutely in the striatum of rats anesthetized with chloral hydrate. Carbon fiber voltammetric microelectrodes were used to monitor extracellular dopamine at three sites near the probes; immediately adjacent to the probe, 220-250 microm from the probe, and 1 mm from the probe. Probes were lowered slowly over a 30 min period, so that dialysate dopamine levels were stable, in the low nanomolar range, and partially TTX-sensitive by the time experiments began. Starting 2h after probe implantation, dopamine was monitored by fast-scan cyclic voltammetry during electrical stimulation of the medial forebrain bundle and during administration of the dopamine uptake inhibitor, nomifensine. The findings of this study show that a gradient of dopamine release and uptake activity extends at least 220 microm from microdialysis probes implanted acutely in the striatum of the anesthetized rat.

Voltammetric study of the control of striatal dopamine release by glutamate.

The central dopamine systems are involved in several aspects of normal brain function and are implicated in a number of human disorders. Hence, it is important to understand the mechanisms that control dopamine release in the brain. The striatum of the rat receives both dopaminergic and glutamatergic projections that synaptically target striatal neurons but not each other. Nevertheless, these afferents do form frequent appositional contacts, which has engendered interest in the question of whether they communicate with each other despite the absence of a direct synaptic connection. In this study, we used voltammetry in conjunction with carbon fiber microelectrodes in anesthetized rats to further examine the effect of the ionotropic glutamate antagonist, kynurenate, on extracellular dopamine levels in the striatum. Intrastriatal infusions of kynurenate decreased extracellular dopamine levels, suggesting that glutamate acts locally within the striatum via ionotropic receptors to regulate the basal extracellular dopamine concentration. Infusion of tetrodotoxin into the medial forebrain bundle or the striatum did not alter the voltammetric response to the intrastriatal kynurenate infusions, suggesting that glutamate receptors control a non-vesicular release process that contributes to the basal extracellular dopamine level. However, systemic administration of the dopamine uptake inhibitor, nomifensine (20 mg/kg i.p.), markedly decreased the amplitude of the response to kynurenate infusions, suggesting that the dopamine transporter mediates non-vesicular dopamine release. Collectively, these findings are consistent with the idea that endogenous glutamate acts locally within the striatum via ionotropic receptors to control a tonic, impulse-independent, transporter-mediated mode of dopamine release. Although numerous prior in vitro studies had suggested that such a process might exist, it has not previously been clearly demonstrated in an in vivo experiment.