Analysis of Protein Synthesis in the Brain Using Cell-Free Techniques

Brown, Ian R.; Cosgrove, James W.

doi:10.1007/978-1-4899-4555-6_1

Cited by 5 publications

(4 citation statements)

References 129 publications

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“…It is possible that an increase in intracellular sodium is most critical for causing inhibition, regardless of energy state (Jones and McIlwain,197 1). Investigations using cell-free synthesis systems prepared from animals following a number of treatments that inhibit protein synthesis still demonstrate a decreased rate of peptide chain initiation, which is associated with polysome disaggregation (Brown and Cosgrove, 1982;Dunlop, 1982). As the mechanisms of ischemic damage and glutamate toxicity become better understood, it may be possible to establish precisely what intracellular events inhibit protein synthesis.…”

Section: Discussionmentioning

confidence: 99%

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

Vornov¹,

Coyle

1991

Journal of Neurochemistry

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In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.

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Section: Discussionmentioning

confidence: 99%

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

Vornov¹,

Coyle

1991

Journal of Neurochemistry

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“…In this study we have examined the abundance of rat brain poly(A')mRNA populations by analysing the molar abundance of newly synthesised radiolabelled protein species on 2-D gels using an exogenous cell-free preparation, a system which reflects biologically active mRNA abundance [3]. The figures presented use newly synthesised actin as standard.…”

Section: In Vitro Translational Analysismentioning

confidence: 99%

“…The high resolution obtained with discontinuous buffer systems such as Laemmli's [l] was further improved with Farrell [2]. The latter procedure makes it possible to separate almost every single protein component of a complex sample, but in most cases, due to the complexity of the pattern obtained, its analysis requires computerized image processing [3]. Other methodological variants in the electrophoretic analysis of proteins make use of a different combination of two electrophoretic methods in order to resolve minor components from complex mixtures.…”

Section: Introductionmentioning

confidence: 99%

An estimation of the sensitivity of in vitro translation using two‐dimensional gel analysis

Perrett

Whatley

1991

Electrophoresis

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Poly (A+ mRNA species, isolated from 100-day-old rat brain, were analysed by in vitro translation and two-dimensional gel electrophoresis. The synthesis of selected protein species was compared to actin on the basis of [35S]methionine incorporation. The estimated molar abundance of translation products varied from abundant species at 0.78% of the total to several are species, detectable below the 0.02% level. If these synthesis rates reflect the abundance of particular mRNAs in the mixture, this sensitivity limit compares well with accepted values using differential cDNA screening techniques. This analysis provides evidence that in vitro translation methodology is able to detect rarer mRNA species than is usually expected--these include similar abundance classes to library screening procedures.

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“…a venous administration of the drug. Most cell-free translation systems derived from brain tissue are unable to engage in initiation (for review, see Brown and Cosgrove, 1983); however, we have developed a system derived from rabbit brain which demonstrates a capacity for initiation in vitro .…”

Section: Fig 2 Binding Of [35s]mentioning

confidence: 99%

Effect of Intravenous Administration of D‐Lysergic Acid Diethylamide on Initiation of Protein Synthesis in a Cell‐Free System Derived from Brain

Cosgrove

Brown

1984

Journal of Neurochemistry

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An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.

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Analysis of Protein Synthesis in the Brain Using Cell-Free Techniques

Cited by 5 publications

References 129 publications

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

An estimation of the sensitivity of in vitro translation using two‐dimensional gel analysis

Effect of Intravenous Administration of D‐Lysergic Acid Diethylamide on Initiation of Protein Synthesis in a Cell‐Free System Derived from Brain

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