Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens. Application to the glyceraldehyde 3-phosphate dehydrogenases from different species

It was previously proposed that an immunological cross-reaction between two denatured proteins is evidence for an homology betweeen their amino sequence (Arnon &; Maron, 1971; Arnheim et al., 1971) and that detection of such a cross-reaction could then be a rapid method to detect sequence homologies (Zakin et al., 1978). In order to test the possibilities of such a methodology, using proteins of known structure, glyceraldehyde 3-phosphate dehydrogenases from different sources are compared by immunochemical techniques. The antibodies raised against the native enzyme from E. coli K 12 can only recognize the homologous antigen, the glyceraldehyde 3-phosphate dehydrogenase from B. stearothermophilus and to a lesser extent that from halibut. In contrast, the antibodies raised against the denatured enzyme from E. coli K 12 can recognize the glyceraldehyde 3-phosphate dehydrogenases from man, ostrich, chicken, sturgeon, halibut, lobster and yeast, when in their denatured state. The present results show unambiguously that through exposure of buried sequences, the immunochemical detection of sequence homologies among proteins is more discriminating when unfolded proteins are used, rather than native ones. It is also proposed that the use of denatured proteins both as immunogens and antigens would be a useful tool in studying biochemical evolution.     

Excitatory amino acids and potassium release in the frog spinal cord

Synaptic release of excitatory amino acids such as L-glutamate and/or L-aspartate and subsequent activation of specific receptors by these putative transmitters appears necessary for the release of K+ by afferent stimulation in the isolated frog spinal cord. This conclusion is based on the findings that (-)baclofen, which is thought to reduce the presynaptic release of putative excitatory amino acid transmitters, and some amino dicarboxylic amino acids (D, L-alpha-aminoadipic acid, 2-amino-4-phosphonobutyric acid, and D, L-alpha, epsilon-diaminopimelic acid), which are believed to interfere with the activation of receptors by these same excitatory amino acids, significantly attenuate the increment in extracellular K+ evoked by tetanic dorsal root stimulation.     

Modification of Redox Sites of N-Methyl-D-Aspartate Receptors Affects Anoxia-Induced Changes in the Bioelectrical Activity of Rat Brain Olfactory Cortex Slices

Living slices of Wistar-Kyoto rat brain olfactory cortex were used to study the effects of the thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which inhibits NMDA receptor activity, on changes in the generation of evoked focal potentials (NMDA and non-NMDA EPSP) in response to long-term and short-term anoxia, which induces functional damage and facilitates increases in the resistance of neurons to severe hypoxia respectively. These studies showed that DTNB (200 'M) efficiently prevented the suppression of focal EPSP generation due to long-term anoxia in most slices. In addition, DTNB partially reversed the protective effect of preconditioning with short-term anoxia on the impairment of focal EPSP generation induced by long-term anoxia. This affected the NMDA component of the EPSP to a greater extent than the non-NMDA component. The possible role of changes in the state of modulatory redox sites of NMDA receptors in the mechanisms of functional damage and increases in neuron resistance due to hypoxia is discussed.     

Comparison of Changes in Glutamate Levels in the Nucleus Accumbens of the Rat Brain during Food Consumption in Conditions of Blockade of Dopamine D1 and D2 Receptors

The effects of blockade of D₁ and D₂ dopamine receptors in the nucleus accumbens on changes in glutamate levels in the intercellular space of this structure during food consumption were studied in Sprague–Dawley rats by intracerebral microdialysis combined with HPLC. These experiments showed that food consumption was accompanied by decreases in glutamate levels in the intercellular spaces of the nucleus accumbens. Blockade of D₁ dopamine receptors with SCH-23390 (0.01 mM) produced no changes in the dynamics of glutamate release during food consumption. Food consumption in conditions of blockade of D₂ dopamine receptors with raclopride (0.01 mM) induced increases in glutamate levels. These data suggest that glutamate levels during food consumption are controlled by the dopaminergic system of the nucleus accumbens, mediated by D₂ but not D₁ dopamine receptors.     

The involvement of glutamatergic transmission in the mechanism of movement disorders induced by reversive rotation of white mice

The ability of the selective non-competitive NMDA receptor blocker MK-801 and a series of new glutamate antagonists—the adamantane derivatives IEM-1754 and IEM-1857 and phencyclidine (IEM-1925)—to prevent movement disorders induced by reversive rotation in mice was studied. I.p. MK-801 at a dose of 0.15 ml and IEM-1754 at a dose of 5.0 mg/kg prevented the development of akinesia in response to reversive rotation, as effectively as scopolamine, a known agent which provides effective prophylaxis for movement diseases. IEM-1857, the quaternary analog of IEM-1754, was not effective. IEM-1925 significantly increased the responses of mice to reversive rotation, possibly because of its high activity in relation to other subtypes of glutamate receptors. These data provide evidence for the involvement of glutamatergic transmission in the mechanism of movement disorders of vestibular origin. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 497–501, May, 1999.     

Caudal end of the rat spinal dorsal horn

We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

一氧化氮对谷氨酸单钠脑损害小鼠学习记忆能力的影响

目的　观察谷氨酸单钠 (MSG)对小鼠学习记忆的影响及血浆、脑内NO含量的变化。方法　给断乳分窝小鼠MSG灌胃 ,每天 2次 ,连续 30d ,31d早灌胃后对小鼠进行迷宫行为训练 ,2 4h后对其进行迷宫记忆检测 ,用硝酸还原酶法检测血浆脑内的NO含量。结果　MSG对小鼠学习记忆能力有影响并存在剂量效应关系 ,其血浆、脑中NO含量均升高并有显著差异 (P <0 .0 5 )。结论　NO可加重MSG对小鼠学习与记忆的损害作用。