Quantitative dual-probe microdialysis: evaluation of [3H]mannitol diffusion in agar and rat striatum

Dual-probe microdialysis was used to study interstitial diffusion in the rat brain. A radiolabelled tracer, (3H]mannitol, was continuously infused at different concentrations via a probe acutely implanted into the striatum of an anaesthetized male rat or into a dilute agar gel. Samples were collected by a second probe placed 1 mm away from the first, and the recovered [3H]mannitol was measured by liquid scintillation counting. In the striatum, the delivery of [3H]mannitol was counteracted by its removal from the extracellular space by passive uptake into cells and clearance into the microcirculation, causing the diffusion profile to approach quasi steady-state levels within 2 h. Diffusion data from brain and agar were analysed using a mathematical model. The apparent (effective) diffusion coefficient for [3H]mannitol was D* = 2.9 x 10(-6) cm2/s, the effective volume fraction alpha* = 0.30 and the clearance rate constant kappa= 2.3 x 10(-5)/s. A tortuosity, lambda = 1.81, and penetration distance r = 4.2 mm, were calculated. We conclude that, using dual-probe microdialysis, parameters reflecting geometric and dynamic tissue properties may be obtained using appropriate mathematical analysis. Quantitative dual-probe microdialysis will be valuable in characterizing interstitial diffusion and the clearance processes underpinning volume transmission in the brain.     

Quantitative dual-probe microdialysis: mathematical model and analysis

Steady-state microdialysis is a widely used technique to monitor the concentration changes and distributions of substances in tissues. To obtain more information about brain tissue properties from microdialysis, a dual-probe approach was applied to infuse and sample the radiotracer, [3H]mannitol, simultaneously both in agar gel and in the rat striatum. Because the molecules released by one probe and collected by the other must diffuse through the interstitial space, the concentration profile exhibits dynamic behavior that permits the assessment of the diffusion characteristics in the brain extracellular space and the clearance characteristics. In this paper a mathematical model for dual-probe microdialysis was developed to study brain interstitial diffusion and clearance processes. Theoretical expressions for the spatial distribution of the infused tracer in the brain extracellular space and the temporal concentration at the probe outlet were derived. A fitting program was developed using the simplex algorithm, which finds local minima of the standard deviations between experiments and theory by adjusting the relevant parameters. The theoretical curves accurately fitted the experimental data and generated realistic diffusion parameters, implying that the mathematical model is capable of predicting the interstitial diffusion behavior of [3H]mannitol and that it will be a valuable quantitative tool in dual-probe microdialysis.     

Determination of Norepinephrine in Microdialysis Samples by Microbore Column Liquid Chromatography with Fluorescence Detection Following Derivatization with Benzylamine

A microbore column liquid chromatographic method is described for the determination of norepinephrine (NE) in microdialysis samples from rat brain. The method is based on precolumn derivatization of NE with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in a highly fluorescent and stable benzoxazole derivative. Typically, a 10-microl sample was mixed with 10 microl derivatization reagent containing 0.45 M Caps buffer (pH 12.0), 0.2 M benzylamine, 10 mM potassium hexacyanoferrate(III), and N,N-dimethylformamide (1:1:1:15, v/v). The derivatization was carried out at 50 degrees C for 20 min. Under these conditions only NE and epinephrine produced high fluorescence yields at excitation and emission wavelengths of 345 and 480 nm, respectively, while fluorescence of other catechols and 5-hydroxyindoles was quenched by 10-100 times. The NE derivative was separated on a reversed-phase column (100 x 1.0 mm i.d., packed with C18 silica, 5 microm) within 10 min with no late eluting peaks. The mobile phase consisted of 40 mM Britton-Robinson buffer (pH 7.5) containing 1 mM didodecyldimethylammonium bromide and acetonitrile (34%, v/v), the flow rate was 40 microl/min. The limit of detection (signal-to-noise ratio of 3) for NE was 90 amol in 10 microl sample injected. Microdialysis samples were collected in 5-min intervals from the probes implanted in the hippocampus, frontal cortex, or hypothalamus of awake rats. The basal extracellular NE levels in the respective areas were 4.7 +/- 0.9, 1.8 +/- 0.3, and 0.8 +/- 0.2 fmol/10 microl (mean +/- SE, n = 7). Perfusion with a Ringer solution containing 100 mM K+ increased hippocampal NE levels by 700%, while NE uptake inhibitors maprotiline and amitriptyline administered orally or subcutaneously increased extracellular NE in the frontal cortex by about 300%. On the other hand, reserpine (5 mg/kg) reduced cortical NE levels by 40% 3 h after the administration. This new fluorescence derivatization method provides better selectivity, sensitivity, and speed for NE determination than the electrochemical detection since no late-eluting compounds such as dopamine, serotonin, and their metabolites are detectable in the chromatograms of the microdialysis samples.     

Conservation and Losses of Non-Coding RNAs in Avian Genomes

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous “losses” of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.     

Ethaselen: a potent mammalian thioredoxin reductase 1 inhibitor and novel organoselenium anticancer agent

Mammalian thioredoxin reductase 1 (TrxR1) is considered to be an important anticancer drug target and to be involved in both carcinogenesis and cancer progression. Here, we report that ethaselen, a novel organoselenium compound with anticancer activity, specifically binds to the unique selenocysteine-cysteine redox pair in the C-terminal active site of mammalian TrxR1. Ethaselen was found to be a potent inhibitor rather than an efficient substrate of mammalian TrxR1. It effectively inhibits wild-type mammalian TrxR1 at submicromolar concentrations with an initial mixed-type inhibition pattern. By using recombinant human TrxR1 variants and human glutathione reductase, we prove that ethaselen specifically targets the C-terminal but not the N-terminal active site of mammalian TrxR1. In A549 human lung cancer cells, ethaselen significantly suppresses cell viability in parallel with direct inhibition of TrxR1 activity. It does not, however, alter either the disulfide-reduction capability of thioredoxin or the activity of glutathione reductase. As a downstream effect of TrxR1 inactivation, ethaselen causes a dose-dependent thioredoxin oxidation and enhances the levels of cellular reactive oxygen species in A549 cells. Thus, we propose ethaselen as the first selenium-containing inhibitor of mammalian TrxR1 and provide evidence that selenium compounds can act as anticancer agents based on mammalian TrxR1 inhibition.     

Determination of the dopamine agonist rotigotine in microdialysates from the rat brain by microbore column liquid chromatography with electrochemical detection

Rotigotine, an investigational dopamine agonist formulated as a patch, is being studied in Parkinson's disease. A microdialysis technique, in combination with microbore column liquid chromatography and electrochemical detection, was developed to monitor rotigotine levels in the brain. Microdialysis probes were inserted into the striata of anesthetized rats, and samples were collected during perfusion with Ringer's solution. Rotigotine was separated using a C18 reversed-phase column. The mobile phase consisted of 50mM Na(2)HPO(4) x 2H(2)O, 2.5 mM sodium octyl sulfonate, and pH 4.5; 35% volume to volume acetonitrile. The flow rate was 30 microl/min, and the potential of the glassy carbon electrode was set to +850 mV. The method allowed monitoring of the time course of brain extracellular rotigotine levels with a detection limit of 1 nM following either intravenous (0.5 mg/kg) or subcutaneous (5.0 mg/kg) rotigotine injection.