Extracellular Neuroactive Amino Acids in the Rat Striatum During Ischaemia: Comparison Between Penumbral Conditions and Ischaemia with Sustained Anoxic Depolarisation

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K⁺ (100 m M ). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K⁺ stimulus did not provoke any release of excitatory amino acids in the 24-h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2- and 24-h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is- chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Aurintricarboxylic Acid Protects Hippocampal Neurons from NMDA-and Ischemia-Induced Toxicity In Vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium-dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia-or excitotoxin-induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA-and ischemia-induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

A SEARCH FOR SOME ANG1OSPERM HORMONES AND THEIR METABOLITES IN CAULERPA PASPALOIDES (CHLOROPHYTA)1

The best available methods were used to search for an-giosperm hormones in the green alga Caulerpa paspa-loides (Bory) Greville. Solvent partitioning of methanol extracts led to acidic ethyl acetate fractions that it were run through high-performance liquid chromatography. Each of the resulting 25 fractions was divided in half so that one half was tested in the dwarf rice or lettuce hypocotyl assays for gibberellins and the other half was methylated and silylated for capillary gas chromatography combined with computerized mass spectrometry. By comparing algal mass spectra with spectra in the extensive library of mass spectra and associated Kovats Retention Indices that MacMillan's Bristol group has amassed, indole-3-acetic acid's presence in the alga was confirmed and dioxindole-3-acetic acid was found. However, despite the presence of several peaks of gibberellin-like activity in the bioassays, no gibberellin or gibberellin metabolite was found in the thousands of full mass spectra examined. The gibberellin-like activity in the acidic ethyl acetate fractions was therefore presumably not from any gibberellin known from vascular plants.     

The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYPI gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.Abbreviations HYP1 and HYP2 S. cerevisiae genes encoding hypusine-containing protein Hyplp and Hyp2p, respectively     

The wall associated lipoteichoic acid ofStreptococcus sanguis

A competitive ELISA is described for the measurement of lipoteichoic acid. The assay was used to determine the wall associated lipoteichoic acid ofStreptococcus sanguis which was found to represent only 2–4% of the phenol extractable content. Extracellular lipoteichoic acid was detected even after exhaustive cell washing. This material was not the result ofde novo synthesis because membrane de-polarization had no effect on the amount detected. Since extracellular lipoteichoic acid interfered with the measurement of cell surface antigen, cells were fixed with glutaraldehyde prior to assay. Lipoteichoic acid was demonstrated on the surface of fixed cells which did not leak antigen. The relevance of fixation used in antigen location studies by electron microscopy of immune-labelled cells is discussed.     

Emulsion liquid membrane extraction of lactic acid from aqueous solutions and fermentation broth

Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n-heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. (c) 1993 John Wiley & Sons, Inc.