cAMP-dependent presynaptic regulation of spontaneous glycinergic IPSCs in mechanically dissociated rat spinal cord neurons.
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Spontaneous miniature glycinergic inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with intact glycinergic presynaptic nerve terminals and evoked IPSCs (eIPSCs) in the slice preparation were investigated using nystatin-perforated patch and conventional whole cell recording modes under the voltage-clamp conditions. Trans-ACPD (tACPD) reversibly reduced the mIPSC frequency without affecting the mean amplitude. The effect was mimicked by a specific metabotropic glutamate receptor (mGluR) II subtype agonist, (2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine (L-CCG-I), and a specific mGluRIII subtype agonist, 2-amino-4-phosphonobutyrate (L-AP4). These inhibitory effects on mIPSC frequency were blocked by the specific antagonists for mGluRII, alpha-methyl-1-(2S, 1'S, 2'S)-2-(carboxycyclo propyl) glycine and (RS)-alpha-cyclopropyl-4-phosphonophenylglycine. In the slice preparation, eIPSC amplitude and mIPSC frequency were decreased reversibly by L-CCG-I (10(-6) M) and L-AP4 (10(-6) M). In K(+)-free or K(+)-free external solution with Ba(2+) and Cs(+), Ca(2+)-free or Cd(2+) external solution, the inhibitory effect of tACPD on mIPSC frequency was unaltered. Forskolin and 8-Br-cAMP significantly increased presynaptic glycine release, and prevented the inhibitory action of tACPD on mIPSC frequency. Sp-cAMP, however, did not prevent the inhibitory action of tACPD on mIPSC frequency. It was concluded that the activation of mGluRs inhibits glycine release by reducing the action of cAMP/PKA pathway. (+info)
Intensity-dependent, rapid activation of presynaptic metabotropic glutamate receptors at a central synapse.
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Synaptic signals from retinal bipolar cells were monitored by measuring EPSCs in ganglion cells voltage-clamped at -70 mV. Spontaneous EPSCs were strongly suppressed by l-2-amino-4-phosphonobutyrate (AP-4), an agonist at group III metabotropic glutamate receptors (mGluRs). Agonists of group I or II mGluRs were ineffective. AP-4 also suppressed ganglion cell EPSCs evoked by bipolar cell stimulation using potassium puffs, sucrose puffs, or zaps of current (0.5-1 microA). In addition, AP-4 suppressed Off EPSCs evoked by dim-light stimuli. This indicates that group III mGluRs mediate a direct suppression of bipolar cell transmitter release. An mGluR antagonist, (RS)-alpha-cyclopropyl-4-phosphonophenylyglycine (CPPG), blocked the action of AP-4. When bipolar cells were weakly stimulated, AP-4 produced a large suppression of the EPSC, but CPPG alone had little effect. Conversely, when bipolar cells were strongly stimulated, CPPG produced an enhancement of the EPSC, but AP-4 alone had little effect. This indicates that endogenous feedback regulates bipolar cell transmitter release and that the dynamic range of the presynaptic metabotropic autoreceptor is similar to that of the postsynaptic ionotropic receptor. Furthermore, the feedback is rapid and intensity-dependent. Hence, concomitant activation of presynaptic and postsynaptic glutamate receptors shapes the responses of ganglion cells. (+info)
Electrophysiological effects of GABA on cat pancreatic neurons.
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In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors. (+info)
Investigations of photoreceptor synaptic transmission and light adaptation in the zebrafish visual mutant nrc.
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PURPOSE: To characterize the retinal physiology of the zebrafish visual mutant no optokinetic response c (nrc) and to identify the genetic map position of the nrc mutation. METHODS: Electroretinograms were recorded from wild-type and nrc zebrafish larvae between 5 to 6 days postfertilization. Responses to flash stimuli, On and Off responses to prolonged light stimuli, and responses to flash stimuli with constant background illumination were characterized. The glutamate agonist, 2-amino-4-phosphonobutyric acid (APB) was used to examine the photoreceptor specific a-wave component of the electroretinogram. Amplified fragment length polymorphism methodology was used to place the nrc mutation on the zebrafish genomic map. RESULTS: nrc and wild-type zebrafish larvae 5 to 6 days postfertilization have similar threshold responses to light, but the b-wave of the nrc electroretinogram is significantly delayed and reduced in amplitude. On and Off responses of nrc larvae to prolonged light have multiple oscillations that do not occur in normal zebrafish larvae after 5 days postfertilization. Analysis of the b-wave demonstrated a light adaptation defect in nrc that causes saturation at background light levels approximately 1 order of magnitude less than those with wild-type larvae. Application of the glutamate analog, APB, uncovered the photoreceptor component of the electroretinogram and revealed a light adaptation defect in nrc photoreceptors. The nrc mutation was placed approximately 0.2 cM from sequence length polymorphism marker Z7504 on linkage group 10. CONCLUSIONS: The zebrafish mutant nrc is a possible model for human retinal disease. nrc has defects in photoreceptor synaptic transmission and light adaptation. The nrc mutant phenotype shows striking similarities with phenotypes of dystrophin glycoprotein complex mutants, including patients with Duchenne/Becker muscular dystrophy. Localization of the nrc mutation now makes it possible to evaluate candidate genes and clone the nrc gene. (+info)
Hydrophobic analogues of the winter flounder 'antifreeze' protein.
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The synthesis, solution conformation and ice-growth inhibition properties of four new analogues of the type I 37-residue winter flounder 'antifreeze' protein are reported. All four analogues contain two extra salt bridges to facilitate comparison of results with previously published data. In two analogues, all four threonine residues in the native polypeptide were mutated to 2-amino butyric acid (an unnatural amino acid) and isoleucine, respectively. The butyric acid analogue was approximately 85% helical at 3 degrees C, modified the shape of ice growth, and exhibited reduced hysteresis compared to the native protein (9% at 4 mM). These results show that the gamma-methyl group of threonine, which is present in the sidechain of 2-amino butyric acid, is not sufficient for activity. The isoleucine analogue, in which the threonine hydroxyl group is replaced by an ethyl group, was 100% helical at 3 degrees C, showed no hysteresis but was able to modify the shape of ice crystal growth. In the third and fourth analogues, mutations of the aspartic acids 1 and 5 to alanine, and asparagines 16 and 27 to leucine in the threonine- and valine-substituted analogues did not affect the helicity of the polypeptides, but removed the ability to inhibit ice growth. (+info)
Expression of bar in the plastid genome confers herbicide resistance.
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Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen. (+info)
Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway.
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Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs. For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site. Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome. All mutations obtained were located in the editing site of valyl-tRNA synthetase. More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine. Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids. Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation. (+info)
Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis.
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Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde. Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis. (+info)