Human γγ-Enolase: Two-Site Immunoradiometric Assay with a Single Monoclonal Antibody

Abstract: A monoclonal antibody (mAb), termed BBS/NC/VI-H14 (H14), that reacts with the human enzyme γγ-enolase was prepared. It was directed against the γ-subunit and did not cross-react with the α- or β-subunit. The mAb H14 can be used for quantitative determination of γγ-enolase in a two-site immunoradiometric assay (two-site IRMA). It is also suitable for immunostaining formalin-fixed tissues. The specific identification of γγ-enolase provided by the two-site IRMA with H14 is discussed in relation to the cellular distribution of this protein.     

Expression of the Neuron-Specific Enolase Gene by Rat Oligodendroglial Cells During Their Differentiation

Abstract: We have examined the regulation of neuron-specific γ-enolase gene (NSE) expression in oligodendrocytes at various steps of their differentiation/maturation. We have demonstrated for the first time that NSE is expressed in oligodendroglial cells in vitro and in vivo, and only at a certain stage of differentiation. A heterogeneity of the γ subunit was observed in cultured oligodendrocytes and the same one was found in adult rat brain. The level of γ mRNA increased when precursor cells differentiated into oligodendrocytes. By contrast, no significant change in α-enolase gene expression was observed. High NSE (γγ and αγ) enolase activity was detected in cultured oligodendrocytes. Treatment with basic fibroblast growth factor, which stimulates the proliferation of oligodendrocyte precursor cells and reversibly blocks their differentiation, resulted in lower αγ- and γγ-enolase activities in these cells, but it enhanced αα-enolase activity slightly. These data indicate that γ-enolase gene expression is associated with the differentiation of the oligodendrocytes and that it is repressed in adult fully mature cells.     

Plasminogen Binds Specifically to α-Enolase on Rat Neuronal Plasma Membrane

Abstract: Plasminogen (PGn) that we identified in microglial-conditioned medium has a neurotrophic factor-like effect on cultured neurons. We have also shown that PGn binds specifically to a protein with a molecular mass of 45 kDa in the neuronal plasma membrane. As a candidate PGn receptor-like molecule on the neuronal surface, this 45-kDa protein was purified from the plasma membrane of embryonic rat brain. Amino acid sequence analysis of polypeptides derived from the cleavage of the protein with cyanogen bromide and V8 protease revealed that the 45-kDa protein is identical to rat α-enolase. In fact, PGn was found to bind to purified rat α-enolase and also to a synthetic peptide (30 residues) that corresponds to the carboxyl terminal region of rat α-enolase. Physical properties of the 45-kDa protein, such as molecular mass, isoelectric point, and the ability to form dimers, are quite similar to those of α-enolase. The 45-kDa PGn-binding protein in the plasma membrane was also recognized by anti-rat α-enolase antibody, and pretreatment with α-enolase antibody markedly diminished the PGn-binding to the plasma membrane. In addition, immunocytochemical staining of the cultured cells under the nonpermeable condition showed that α-enolase is present on the cell surface of a certain population of neurons. These results suggest that α-enolase may function as a PGn-binding molecule on the neuronal cell surface.     

GABAA Receptors Containing the α4-Subunit: Prevalence, Distribution, Pharmacology, and Subunit Architecture In Situ

Abstract: Recombinant GABA_A receptors, expressed from α-, β-, and γ2-subunits, are diazepam-insensitive when the α-subunit is either α4 or α6. In situ, diazepam-insensitive receptors containing the α6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABA_A receptors in various brain areas containing the α4-subunit. GABA_A receptors immunoprecipitated with a newly developed α4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2-recombinant receptors and diazepam-insensitive [³H]Ro 15-4513 binding sites in rat brain membranes. In addition, α4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4-subunit containing receptors revealed that the α4-subunit contributes to several receptor subtypes. Depending on the brain region, the α4-subunit can be coassembled with a second type of α4-subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [³H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

The Extraneural Distribution of γ-Hydroxybutyrate

Abstract— γ-Hydroxybutyrate has been found to be widely distributed in both neural and extraneural tissues in the rat. The kidney and brown fat have more than 10 times higher concentrations of y-hydroxybutyrate than does the brain. This observation suggests that γ-hydroxybutyrate may participate in the metabolism of many organs, and that GABA may not be the precursor in extraneural tissues.     

Endogenous γ-Hydroxybutyrate in Rat Brain Areas: Postmortem Changes and Effects of Drugs Interfering with γ-Aminobutyric Acid Metabolism

Abstract: The possibility that γ-hydroxybutyrate (GHB), a metabolite of γ-aminobutyric acid (GABA), may play a role in the CNS has recently come to attention. We describe here a sensitive and specific mass fragmento-graphic technique that allows the measurement of picomole amounts of GHB in single rat brain areas. Moreover, we show that GHB can accumulate postmortem, an effect that is blocked by the use of microwave irradiation to kill the animals. To understand further the relationship between GABA and GHB formation, we treated rats with drugs known to inferfere with GABA metabolism at different levels and concomitantly measured GABA and GHB in cerebral cortex and cerebellum. Isoniazide, which blocks the formation of GABA, also decreases GHB. Blockers of the catabolism of GABA, such as aminooxyacetic acid and γ-acetylenic GABA, increase GABA levels and decrease those of GHB. Sodium dipropylacetate increases both GABA and GHB, supporting the hypothesis that this effective antiepileptic drug also blocks in vivo the enzyme that converts succinic semialdehyde to succinic acid.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Structure of the C-Terminal Tail of α-Tubulin: Increase of Heterogeneity from Newborn to Adult

Abstract: A combination of posttranslational modifications contributes to the high heterogeneity of brain tubulin in mammals. In this report, the structures of the detyrosinated carboxy-terminal peptides of α-tubulin from newborn and adult mouse brain were compared. The heterogeneity of these carboxy-terminal peptides was observed to increase from newborn to adult brain tubulin. The major part of this increased heterogeneity is due to the posttranslational excision of Glu⁴⁵⁰, which makes α-tubulin nontyrosinatable (Δ-2 tubulin). The structures of the polyglutamyl side chain of the bi- and triglutamylated peptides were analyzed in this work. In polyglutamylation of α-tubulin, the first glutamyl residue can only be amide-linked to the γ-carboxyl group of Glu⁴⁴⁵, but the additional residues may be linked either to the γ- or to the α-carboxyl groups of the preceding one. By optimized reverse-phase separations and comparison with synthetic peptides corresponding to all possible linkages for the biglutamylated (γ1α2, γ1γ2) and triglutamylated (γ1α2α3, γ1γ2γ3, γ1α2γ3, γ1γ2α3, γ1γ2α2) tubulin peptides, it was possible to conclude that the mode of linkage connecting the second and third additional glutamyl residues corresponds mostly to α-bond structures, for both newborn and adult mice.     

Polyclonal Antibodies Directed Against an Epitope Specific for the α4-Subunit of GABAA Receptors Identify a 67-kDa Protein in Rat Brain Membranes

Abstract: Polyclonal antibodies were raised to the C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α4-subunit. These anti-peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti-peptide α4 (517–523) antibodies and could then be identified by the anti-α4-antibodies as well as by the GABA_A receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the α4-subunit of GABA_A receptors. Intact GABA_A receptors appeared to be retained by the immunoaffinity column because other GABA_A receptor subunit proteins like the β2/β3-subunits and the γ2-subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67-kDa protein, a 51-kDa protein could be detected by the antibody bd-28 and the anti-peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1-subunit-specific anti-peptide α1 (1–9) antibody. In contrast to the α1-subunit, the 51-kDa protein identified by the anti-α4 antibody could not be deglycosylated by N -Glycanase. The identity of the 51-kDa protein identified by the anti-α4-antibodies thus must be further investigated.     

Neuropeptide γ-(l-9)-Peptide: A Major Product of the Posttranslational Processing of γ-Preprotachykinin in Rat Tissues

Abstract: γ-Preprotachykinin mRNA is the most abundant tachykinin mRNA in rat tissues, but the pathway of posttranslational processing of its translation product is unknown. An antiserum was raised against the synthetic peptide Asp-Ala-Gly-His-Gly-Gln-lle-Ser-His [neuropeptide γ-(1-9)-peptide, equivalent to γ-preprotachykinin-(72-80)-peptide], that showed <1% reactivity with intact neuropeptide γ and other tachykinins. Neuropeptide γ-(1-9)-peptide was detected by radioimmunoassay in relatively high concentrations in extracts of regions of rat brain and gastrointestinal tract. These concentrations correlated with (r = 0.99), but were significantly (p < 0.05) less than, the concentrations of neurokinin A-like immunoreactivity. The neuropeptide γ-(1-9)-like immunoreactivity in an extract of rat brain was eluted from a reverse-phase HPLC column in a single fraction with the same retention time as synthetic neuropeptide γ-(1 -9)-peptide. The synthetic peptide did not contract or relax isolated rat trachea, superior mesenteric artery, stomach fundus, or ileum, and the peptide did not affect the ability of neuropeptide 7 to contract the rat fundus. It is concluded that, in rat tissues, Lys⁷⁰-Arg⁷¹ in 7-preprotachykinin is a major site of posttranslational processing, but the resulting product, neuropeptide γ-(1-9)-peptide, is neither an agonist nor an antagonist at the neurokinin-2 (NK-2) receptor.     

Small N-Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ-aminobutyric acid type A (GABA_A) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)-amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (α6S) and long (wild-type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABA_A receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [³H]muscimol and [³H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit-containing receptors.     

Presence of γ-Aminobutyric Acid in Rat Ovary

Abstract: As γ-aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non-neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.     

Pretreatment of Astrocytes with Interferon-α/β Impairs Interferon-γ Induction of Nitric Oxide Synthase

Abstract: Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Exposure of astrocytes, in primary culture, to interferon-γ results in stimulation of nitric oxide synthase activity and increased nitric oxide release. In contrast to interferon-γ, interferon-α/β had a minimal effect on astrocytic nitric oxide formation. Furthermore, pretreatment of astrocytes with interferon-α/β inhibited (∼65%) stimulation by interferon-γ of nitric oxide synthase activity and nitric oxide release. Treatment with interferon-α/β at a concentration as low as 10 U/ml caused inhibition of mitochondrial cytochrome c oxidase. Furthermore, the damage to cytochrome c oxidase was prevented by the putative interferon-α/β receptor antagonist oxyphenylbutazone. In view of these observations, our current hypothesis is that the mitochondrial damage caused by exposure to interferon-α/β may impair the ability of astrocytes to induce nitric oxide synthase activity on subsequent interferon-γ exposure. These results may have implications for our understanding of the mechanisms responsible for the therapeutic effects of interferon-α/β preparations in multiple sclerosis.     

Distribution of γ-Tubulin in Higher Plant Cells: Cytosolic γ-Tubulin is Part of High Molecular Weight Complexes

Abstract: γ-Tubulin is a protein found in all eukaryotic cells, where it plays a key role in the nucleation of microtubules. In higher plant cells, γ-tubulin is localized at the nuclear surface, a known microtubule-organizing centre, and is codistributed with all microtubule arrays. Functions of plant γ-tubulin remain to be determined. This study describes some properties of higher plant γ-tubulin. The overall level of γ-tubulin was constant during the cell cycle in synchronized tobacco BY-2 cells. Biochemical analysis of the subcellular distribution of γ-tubulin in maize cells revealed that, in contrast with animal γ-tubulin, plant γ-tubulin is mainly associated with endomembranes. We showed for the first time that the pool of soluble cytosolic γ-tubulin contained two main γ-tubulin complexes. γ-tubulin, Hsp70 and TCP1-related proteins might interact in a small complex of 750 kDa. A second γ-tubulin complex, larger than 1500 kDa was purified. The protein profile of this large complex was very similar to animal γ-tubulin complexes. The putative functions of these two complexes in plant microtubule nucleation are discussed.     

Repeated Administration of γ-VinylGABA Reduces Rat Brain Glutamine Synthetase Activity

Abstract: The glutamine cycle has been proposed as a pathway in which glutamine synthesized in glia provides substrate for synthesis of the neurotransmitters glutamate and GABA as they are lost from neurons. To test whether GABA may regulate this pathway, the effect of elevated GABA on the glial enzyme glutamine synthetase was examined in rat brain. Repeated subcutaneous injections of the antiepileptic GABA transaminase inhibitor γ-vinylGABA at a dose of 150 mg/kg per day for 21 days reduced glutamine synthetase activity by 36% in the cortex and 22% in the cerebellum. At 30 mg/kg per day, glutamine synthetase activity was reduced by 9.5% in the cortex but unchanged in the cerebellum. The reductions were brain specific because the skeletal muscle and liver enzymes were unaffected by γ-vinylGABA administration. Amino acid analysis of the cortex from γ-vinylGABA-treated rats demonstrated a 270% increase in GABA levels after 150 mg/kg but no change after 30 mg/kg. GABA levels and glutamine synthetase activity were inversely correlated. The 150 mg/kg dose significantly lowered cortical glutamine and glutamate levels. The decline in brain glutamine synthetase activity with chronic γ-vinylGABA administration developed gradually over time and may be due to the slow turnover of this enzyme in vivo.     

Distribution of mRNAs Encoding the Peroxisome Proliferator-Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System

Abstract: We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.     

The Effect of γ-Aminobutyric Acid on Substrate-Level Phosphorylation in Brain Mitochondria

Abstract: A consequence of the metabolism of γ-aminobutyric acid (GABA) via the "GABA shunt" should be a decreased rate of substrate-level phos- phorylation of GDP to GTP. ³²P₁ labeling of nucleotides was, therefore, studied in uncoupled brain mitochondria with α-ketoglutarate or a-ketoglutarate + GABA as substrates. The addition of an equimolar amount of GABA resulted in an approximately 50% reduction of the final specific activity of all mitochondrial nucleotides. This effect was completely reversed by aminooxyacetic acid. GABA did not affect the time course of nucleotide labeling. Although delineation of the mechanism involved requires further study, these preliminary results suggest an important modulatory role of GABA in the intermediary metabolism of brain mitochondria.