Physiological evidence for ionotropic and metabotropic glutamate receptors in rat taste cells.
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Monosodium glutamate (MSG) elicits a unique taste in humans called umami. Recent molecular studies suggest that glutamate receptors similar to those in brain are present in taste cells, but their precise role in taste transduction remains to be elucidated. We used giga-seal whole cell recording to examine the effects of MSG and glutamate receptor agonists on membrane properties of taste cells from rat fungiform papillae. MSG (1 mM) induced three subsets of responses in cells voltage-clamped at -80 mV: a decrease in holding current (subset I), an increase in holding current (subset II), and a biphasic response consisting of an increase, followed by a decrease in holding current (subset III). Most subset II glutamate responses were mimicked by the ionotropic glutamate receptor (iGluR) agonist N-methyl-D-aspartate (NMDA). The current was potentiated by glycine and was suppressed by the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5). The group III metabotropic glutamate receptor (mGluR) agonist L-2-amino-4-phosphonobutyric acid (L-AP4) usually mimicked the subset I glutamate response. This hyperpolarizing response was suppressed by the mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) and by 8-bromo-cAMP, suggesting a role for cAMP in the transduction pathway. In a small subset of taste cells, L-AP4 elicited an increase in holding current, resulting in taste cell depolarization under current clamp. Taken together, our results suggest that NMDA-like receptors and at least two types of group III mGluRs are present in taste receptor cells, and these may be coactivated by MSG. Further studies are required to determine which receptors are located on the apical membrane and how they contribute to the umami taste. (+info)
Mutational analysis of Asp51 of Anabaena azollae glutamine synthetase. D51E mutation confers resistance to the active site inhibitors L-methionine-DL-sulfoximine and phosphinothricin.
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The role of Asp51 in the catalytic activity of glutamine synthetase from the cyanobacterium Anabaena azollae has been analyzed. Five mutant enzymes, D51S, D51A, D51E, D51N and D51R, were constructed by site-directed mutagenesis and characterized. Asp51 appears to participate in the binding of ammonium ion, as affinity for this substrate was affected in all cases, although it varied according to the charge and/or size of the amino-acid residue, decreasing in the order Glu > Asn > Ser > Ala. The replacement of Asp51 by Glu (D51E) conferred besides a high resistance to the herbicides L-methionine-DL-sulfoximine and phosphinothricin, as a result of a decreased phosphorylation ability. (+info)
Human melanoma-associated retinopathy (MAR) antibodies alter the retinal ON-response of the monkey ERG in vivo.
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PURPOSE: Melanoma-associated retinopathy (MAR) is a paraneoplastic condition that causes visual symptoms of night-blindness and photopsias. The electroretinogram (ERG) of MAR patients is characteristically abnormal in a way that implicates retinal depolarizing bipolar cell (DBC) dysfunction. Whether an injection of IgG from MAR patients into the vitreous of monkeys would alter the ERG acutely as a demonstration of a functional basis for patients' visual symptoms was explored. METHODS: MAR IgG was isolated from three visually symptomatic melanoma patients. Control IgG was from melanoma patients with no vision problems. The ERG was monitored after intravitreal injections into monkey eyes. One eye was injected with 2-amino-4-phosphonobutyric acid (APB), which is known to block DBC ON-pathway responses. Retinal immunocytochemistry was performed using fluorescein isothiocyanate-labeled goat anti-human IgG. RESULTS: Within 1 to 3 hours after MAR IgG injection, the ERG photopic b-wave was diminished, with far less effect on the a- and d-waves. These changes are characteristic of DBC dysfunction and were similar to the effects of APB. The scotopic ERG b-wave, which reflects activity of rod-driven DBCs, showed a loss of amplitude and threshold sensitivity after MAR IgG. Retinal immunocytochemistry with anti-IgG antibody showed IgG penetration throughout the retinal layers, but staining was not specific for a single type of retinal neuron. CONCLUSIONS: Intravitreal injection of human MAR IgG altered the monkey ERG acutely in ways that implicate functional disruption of retinal DBC signaling. These results support the hypothesis that MAR IgG circulating antibodies are responsible for the reported visual symptoms. Bipolar cells in the ON-pathway appear to be affected more than OFF-pathway bipolar cells of the cone pathway in this acute preparation. (+info)
In vivo antitumor activity and induction of insulin-like growth factor-1-resistant apoptosis by SC-alphaalphadelta9.
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We previously showed that SC-alphaalphadelta9 (4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid) is a novel antiphosphatase agent that selectively inhibits the growth of transformed cells in culture and affects elements of insulin-like growth factor-1 (IGF-1) signaling. We now show that SC-alphaalphadelta9 induces IGF-1-resistant apoptosis and kills tumor cells in vivo. In cultured murine 32D cells, SC-alphaalphadelta9 induced concentration-dependent apoptosis that was blocked by ectopic Bcl-2 expression. No apoptosis was detected in 32D cells treated with the congener SC-alpha109, which lacks the ability to disrupt IGF-1 signaling. After interleukin-3 withdrawal or etoposide treatment, exogenous IGF-1 prevented apoptosis and elevated levels of Cdc2, a biochemical indicator of a functional IGF-1 receptor pathway. In contrast, exogenous IGF-1 did not prevent apoptosis or loss of Cdc2 expression caused by SC-alphaalphadelta9. Furthermore, IGF-1 receptor overexpression failed to protect cells against SC-alphaalphadelta9-induced apoptosis. Kinetic analyses demonstrated that Cdc2 down-regulation after SC-alphaalphadelta9 treatment preceded both apoptosis and loss of the IGF-1 receptor, indicating that loss of Cdc2 was a direct effect of SC-alphaalphadelta9 treatment and not secondary to cell death. IGF-1 receptor autophosphorylation studies indicated that SC-alphaalphadelta9 did not interact directly with the IGF-1 receptor nor bind to the growth factor itself, suggesting a site of action distal to the IGF-1 receptor. In the SCCVII murine tumor model, a single i.p. injection of SC-alphaalphadelta9 caused a dose-dependent decrease in clonogenic cell survival. The IC(50) of SC-alphaalphadelta9 was 35 mg/kg, comparable to 25 mg/kg carboplatin. The ability to induce IGF-1-resistant apoptosis distinguishes SC-alphaalphadelta9 from other apoptosis-inducing agents and suggests compounds of this class deserve further study as potential anticancer agents. (+info)
Activity and calcium-dependent mechanisms maintain reliable interneuron synaptic transmission in a rhythmic neural network.
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Inputs from glutamatergic excitatory interneurons (EIN) to motor neurons in the lamprey spinal cord locomotor network exhibit activity-dependent depression during spike trains. The mechanism underlying this depression has been examined here, and its relevance to transmitter release during rhythmic activity has been investigated. The depression of EIN inputs was greater after larger initial EPSPs and reduced in low-calcium Ringer's solution, effects that are consistent with depression caused by depletion of releasable transmitter stores. However, the depression was greater at lower stimulation frequencies and could be reversed by increasing the stimulation frequency. In addition, high-calcium Ringer's solution and the slow intracellular calcium chelator EGTA-AM, which both failed to affect the amplitude of low frequency-evoked EPSPs, reduced and increased the depression, respectively. These results are inconsistent with a simple depletion mechanism but suggest that ongoing activity and calcium-dependent mechanisms oppose depletion. The network relevance of this mechanism was examined using physiologically relevant bursts to simulate EIN spiking during rhythmic activity. Although considerably more EPSPs were evoked than during spike trains, burst-evoked EPSPs did not depress. However, single EPSPs evoked at the interburst interval depressed, and burst transmission was disrupted by EGTA-AM, again suggesting the involvement of activity and calcium-dependent mechanisms. By responding to the calcium changes evoked by increased interneuron activity, this mechanism can monitor transmitter requirements caused by EIN spiking, allowing reliable transmission across different patterns of network activity. However, not all types of spinal interneurons exhibit reliable burst transmission, suggesting specificity of this mechanism to a subset of neurons. (+info)
Cortical cell orientation selectivity fails to develop in the absence of ON-center retinal ganglion cell activity.
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Neuronal activity is necessary for the normal development of visual cortical cell receptive fields. When neuronal activity is blocked, cortical cells fail to develop normal ocular dominance and orientation selectivity. Patterned activity has been shown to play an instructive, rather than merely permissive, role in the segregation of geniculocortical afferents into ocular dominance columns. To test whether normal patterns of activity are necessary to instruct the development of cortical orientation selectivity, we studied ferrets raised without ON-center retinal ganglion cell activity. The ON-center blockade was produced by daily intravitreal injections of DL-2-amino-4-phosphonobutyric acid (APB). Effects of this treatment on the development of orientation selectivity in primary visual cortex were assessed using extracellular electrode recordings and optical imaging. In animals raised with an ON-center blockade starting after visual cortical cells are visually driven but still poorly tuned for orientation, cortical cell responsivity was maintained, but no maturation of orientation selectivity was seen. No recovery of orientation tuning was seen in animals treated with APB during the normal period of orientation development and then allowed several months of development without treatment. These results suggest that patterns of neuronal activity carried in the separate ON- and OFF-center visual pathways are necessary for the development of orientation selectivity in visual cortical neurons of the ferret and that there is a critical period for this development. (+info)
Development of a nonantibiotic dominant marker for positively selecting expression plasmids in multivalent Salmonella vaccines.
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We report the novel application of a herbicide-resistance-based dominant marker for the positive selection of expression plasmids in Salmonella serovar vaccines. The beta-lactamase gene of the plasmid pTETnir15, which expresses fragment C of tetanus toxin (TetC), has been replaced with the bar gene marker. The new plasmid pBAT1 can be positively selected in vitro within Salmonella serovars in the presence of the herbicide DL-phosphinothricin. The expression of TetC remains unaltered, and the Salmonella enterica serovar Typhimurium vaccine strain is stable and immunogenic in vivo. (+info)
Light evokes Ca2+ spikes in the axon terminal of a retinal bipolar cell.
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Bipolar cells in the vertebrate retina have been characterized as nonspiking interneurons. Using patch-clamp recordings from goldfish retinal slices, we find, however, that the morphologically well-defined Mb1 bipolar cell is capable of generating spikes. Surprisingly, in dark-adapted retina, spikes were reliably evoked by light flashes and had a long (1-2 s) refractory period. In light-adapted retina, most Mb1 cells did not spike. However, an L-type Ca2+ channel agonist could induce periodic spiking in these cells. Spikes were determined to be Ca2+ action potentials triggered at the axon terminal and were abolished by 2-amino-4-phosphonobutyric acid (APB), an agonist that mimics glutamate. Signaling via spikes in a specific class of bipolar cells may serve to accelerate and amplify small photo-receptor signals, thereby securing the synaptic transmission of dim and rapidly changing visual input. (+info)