ABSTRACT
A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method has been validated for determining concentrations of glutamate, glycine, and alanine in human plasma. Proteins in plasma were precipitated with perchloric acid, followed by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Simultaneous analysis of glutamate, glycine, and alanine is achieved using reversed-phase HPLC conditions and ultraviolet detection. Excellent linearity was observed for these three amino acids over their concentration ranges with correlation coefficients (r)>0.999. The intra- and inter-day precision were below 10%. This method utilizes quality control samples and demonstrates excellent plasma recovery and accuracy. The developed method has been successfully applied to measure plasma glutamate, glycine, and alanine in twenty volunteers.
Subject(s)
Humans , Alanine , Amino Acids , Aminoquinolines , Carbamates , Chromatography, High Pressure Liquid , Chromatography, Liquid , Glutamic Acid , Glycine , Perchlorates , Plasma , Proteins , Quality ControlABSTRACT
The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20+/-0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26+/-0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32+/-0.03. The rectification index (control 2.39+/-0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02+/-0.11 and 0.93+/-0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.
Subject(s)
Animals , Rats , 6-Cyano-7-nitroquinoxaline-2,3-dione , Adamantane , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid , Excitatory Postsynaptic Potentials , N-Methylaspartate , Piperidines , Plastics , Receptors, AMPA , Receptors, Glutamate , Receptors, N-Methyl-D-Aspartate , Synaptic Transmission , ThalamusABSTRACT
The effect of forskolin on corticostriatal synaptic transmission was examined by recording excitatory postsynaptic currents (EPSCs) in rat brain slices using the whole-cell voltage-clamp technique. Forskolin produced a dose-dependent increase of corticostriatal EPSCs (1, 3, 10, and 30micrometer) immediately after its treatment, and the increase at 10 and 30micrometer was maintained even after its washout. When the brain slices were pre-treated with (DL)-2-amino-5-phosphonovaleric acid (AP-V, 100micrometer), an NMDA receptor antagonist, the acute effect of forskolin (10micrometer) was blocked. However, after washout of forskolin, an increase of corticostriatal EPSCs was still observed even in the presence of AP-V. When KT 5720 (5micrometer), a protein kinase A (PKA) inhibitor, was applied through the patch pipette, forskolin (10micrometer) increased corticostriatal EPSCs, but this increase was not maintained. When forskolin was applied together with AP-V and KT 5720, both the increase and maintenance of the corticostriatal EPSCs were blocked. These results suggest that forskolin activates both NMDA receptors and PKA, however, in a different manner.
Subject(s)
Animals , Rats , Brain , Carbazoles , Cyclic AMP-Dependent Protein Kinases , Excitatory Postsynaptic Potentials , Colforsin , N-Methylaspartate , Patch-Clamp Techniques , Pyrroles , Receptors, N-Methyl-D-Aspartate , Synaptic TransmissionABSTRACT
The strain of DG7 isolated from the oilwater sample in DAGANG oilfield could produce surfactant ?acids and gas in the culture medium in which the diesel was sole carbon source .The strain was Gram-negative, motive, rod and growed facultatively in the 0 to 18.5% NaCl. Based on its characters, the strain was identified as a member of the genus Aeromonas, but there were some differences between this strain and the described species of this genus in some biochemical features, suggesting that it could be a new species of the genus.