Molecular cloning and characterization of cDNA encoding the alpha subunit of the rat protein synthesis initiation factor eIF-2B.

Eukaryotic initiation factor 2B (eIF-2B) is an essential component of the pathway of peptide-chain initiation in mammalian cells, yet little is known about its molecular structure and regulation. To investigate the structure, regulation, and interactions of the individual subunits of eIF-2B, we have begun to clone, characterize, and express the corresponding cDNAs. We report here the cloning and characterization of a 1510-bp cDNA encoding the alpha subunit of eIF-2B from a rat brain cDNA library. The cDNA contains an open reading frame of 918 bp encoding a polypeptide of 305 aa with a predicted molecular mass of 33.7 kDa. This cDNA recognizes a single RNA species approximately 1.6 kb in length on Northern blots of RNA from rat liver. The predicted amino acid sequence contains regions identical to the sequences of peptides derived from bovine liver eIF-2B alpha subunit. Expression of this cDNA in vitro yields a peptide which comigrates with natural eIF-2B alpha in SDS/polyacrylamide gels. The predicted amino acid sequence exhibits 42% identity to that deduced for the Saccharomyces cerevisiae GCN3 protein, the smallest subunit of yeast eIF-2B. In addition, expression of the rat cDNA in yeast functionally complements a gcn3 deletion for the inability to induce histidine biosynthetic genes under the control of GCN4. These results strongly support the hypothesis that mammalian eIF-2 alpha and GCN3 are homologues. Southern blots indicate that the eIF-2B alpha cDNA also recognizes genomic DNA fragments from several other species, suggesting significant homology between the rat eIF-2B alpha gene and that from other species.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

Metabolomics analysis in rats with thiamine deficiency identifies key metabolites in vulnerable brain regions and suggests neural stem progenitor cells play a role in ameliorating metabolic dysfunction

La documentation scientifique fait état de la présence, chez l’adulte, de cellules souches et progénitrices neurales (CSPN) endogènes dans les zones sous-ventriculaire et sous-granulaire du cerveau ainsi que dans le gyrus denté de l’hippocampe. De plus, un postulat selon lequel il serait également possible de retrouver ce type de cellules dans la moelle épinière et le néocortex des mammifères adultes a été énoncé. L’encéphalopathie de Wernicke, un trouble neurologique grave toutefois réversible qui entraîne un dysfonctionnement, voire une défaillance du cerveau, est causée principalement par une carence importante en thiamine (CT). Des observations récentes laissent envisager que les facteurs en cause dans la prolifération et la différenciation des CSPN pourraient également jouer un rôle important lors d’un épisode de CT. L’hypothèse, selon laquelle l’identification de nouveaux métabolites entrant dans le mécanisme ou la séquence de réactions se soldant en une CT pourraient en faciliter la compréhension, a été émise au moyen d'une démarche en cours permettant d’établir le profil des modifications métaboliques qui surviennent en de telles situations. Cette approche a été utilisée pour constater les changements métaboliques survenus au niveau du foyer cérébral dans un modèle de rats déficients en thiamine (rats DT), particulièrement au niveau du thalamus et du colliculus inférieur (CI). La greffe de CSPN a quant à elle été envisagée afin d’apporter de nouvelles informations sur la participation des CSPN lors d’un épisode de CT et de déterminer les bénéfices thérapeutiques potentiels offerts par cette intervention. Les sujets de l’étude étaient répartis en quatre groupes expérimentaux : un premier groupe constitué de rats dont la CT était induite par la pyrithiamine (rats DTiP), un deuxième groupe constitué de rats-contrôles nourris ensemble (« pair-fed control rats » ou rats PFC) ainsi que deux groupes de rats ayant subi une greffe de CSPN, soit un groupe de rats DTiP greffés et un dernier groupe constitué de rats-contrôles (rats PFC) greffés. Les échantillons de foyers cérébraux (thalamus et CI) des quatre groupes de rats ont été prélevés et soumis à des analyses métabolomiques non ciblées ainsi qu’à une analyse visuelle par microscopie à balayage électronique (SEM). Une variété de métabolites-clés a été observée chez les groupes de rats déficients en thiamine (rats DTiP) en plus de plusieurs métabolites dont la documentation ne faisait pas mention. On a notamment constaté la présence d’acides biliaires, d’acide cynurénique et d’acide 1,9— diméthylurique dans le thalamus, alors que la présence de taurine et de carnosine a été observée dans le colliculus inférieur. L’étude a de plus démontré une possible implication des CSPN endogènes dans les foyers cérébraux du thalamus et du colliculus inférieur en identifiant les métabolites-clés ciblant les CSPN. Enfin, les analyses par SEM ont montré une amélioration notable des tissus à la suite de la greffe de CSPN. Ces constatations suggèrent que l’utilisation de CSPN pourrait s’avérer une avenue thérapeutique intéressante pour soulager la dégénérescence symptomatique liée à une grave carence en thiamine chez l’humain.

GABAA receptor beta subunit mRNA expression in the human alcoholic brain

A competitive RT-PCR assay was used to quantify the expression of the GABA(A) receptor beta(1), beta(2) and beta(3) isoform mRNA transcripts in the superior frontal cortex and motor cortex of 21 control and 22 alcoholic cases. A single set of primers was designed that permitted amplification of all three transcripts and the internal standard simultaneously; differentiation of the individual transcripts was achieved by restriction enzyme digestion. Construction of a standard curve, using the internal standard and a concentration range of beta(2) cRNA-enabled quantitation of mRNA expression levels. No significant difference in mRNA expression was found between the control and alcoholic case groups in either the superior frontal or motor cortex for the beta(2) or beta(3) isoforms. A significant interaction was found between isoform and area, although, the two case groups did not partition on this measure. The interaction was due to a significant difference between superior frontal and motor cortex for the beta(3) isoform; this regional comparison was not significant for beta(2) mRNA. Age at death and post-mortem delay (PMD) had no significant effect on beta mRNA expression in either case group in either region. A beta(1) signal could not be detected in the RT-PCR assay. (C) 2004 Elsevier Ltd. All rights reserved.

Genetic study of alcoholism and novel gene expression in the alcoholic brain

Alcohol dependence may result from neuroadaptation involving alteration of gene expression after long-term alcohol exposure. The systematic study of gene expression profiles of the human alcoholic brain was initiated using the method of polymerase chain reaction (PCR)-differential display and was followed by DNA microarray. To date, more than 100 alcohol-responsive genes have been identified from the frontal cortex, motor cortex and nucleus accumbens of the human brain. These genes have a wide range of functions in the brain and indicate diverse actions of alcohol on neuronal function. This review discusses the current information on the genetic basis of alcoholism and the induction and characterization of these alcohol-responsive genes.

Loss of glial glutamate transporters and induction of neuronal expression of GLT-1B in the hypoxic neonatal pig brain

The homeostasis of glutamate is critical to normal brain function; deficiencies in the regulation of extracellular glutamate are thought to be a major determinant of damage in hypoxic brains. Extracellular levels of glutamate are regulated mainly by plasmalemmal glutamate transporters. We have evaluated the distribution of the glutamate transporter GLAST and two splice variants of GLT-1 in the hypoxic neonatal pig brain using this as model of neonatal humans. In response to severe hypoxic insults, we observe a rapid loss of two glial glutamate transporters from specific brain regions, such as the CA1 region of the hippocampus, but not the dentate gyrus. The spatial distribution of loss accords with patterns of damage in these brains. Conversely, we demonstrate that hypoxia evokes the expression of a splice variant of GLT-1 in neurons. We suggest that this expression may be induced in response to elevated extracellular glutamate around these neurons, and that this splice variant may represent a useful marker for direct quantification of the extent of likely neuronal damage in hypoxic brains. © 2004 Elsevier B.V. All rights reserved.

The pathophysiology of 'brain shrinkage' in alcoholics - Structural and molecular changes and clinical implications

This article represents a symposium of the 2004 ISBRA Congress held in Mannheim. The presentations were: Review of the neuropathological and neurochemical changes seen in alcohol-related ' brain shrinkage ' by Clive Harper; In Vivo Detection of Macrostructural and Microstructural Markers of Brain Integrity in Human Alcoholism and a Rodent Model of Alcoholism by Adolf Pfefferbaum, Elfar Adalsteinsson and Edith Sullivan; Gene and Protein Changes in the Brains of Alcoholics with ' Brain Shrinkage ' by Joanne Lewohl and Peter Dodd; Cross sectional and longitudinal MR spectroscopy studies of chronic adult alcoholics by Michael Taylor; Brain Atrophy Associated with Impairment on a Simulated Gambling Task in Long-Term Abstinent Alcoholics by George Fein and Bennett Landman.

Comparative gene expression in brain regions of human alcoholics

The mesocorticolimbic system is the reward centre of the brain and the major target for drugs of abuse including alcohol. Neuroadaptive changes in this region are thought to underlie the process of tolerance and dependence. Recently, several research groups have searched for alcohol-responsive genes using high-throughput microarrays and well-characterized human post-mortem material. Comparison of data from these studies of cortical regions highlights the differences in experimental approach and selection of cases. However, alcohol-responsive gene sets associated with transcription, oxidative stress and energy production were common to these studies. In marked contrast, alcohol-responsive genes in the nucleus accumbens and the ventral tegmental area are primarily associated with changes in neurotransmission and signal transduction. These data support the concept that, within cortical regions, changes in gene expression are associated with alcoholism-related pathology. In the dopaminergic tract of the mesocorticolimbic system, alcohol-responsive gene sets suggest long-term neuroplastic changes in synaptic transmission.

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 ( unit-cell parameters a = b = 136.83, c = 99.82 angstrom, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 angstrom resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Identification of a novel inositol phosphate recognition site: specific [3H]inositol hexakisphosphate binding to brain regions and cerebellar membranes

[3H]Inositol hexakisphosphate (InsP6) binds with a heterogeneous distribution to frozen sections of unfixed rat brain and is displaced by unlabelled InsP6. The pattern of binding correlates with binding to neuronal cell bodies. [3H]InsP6 binding to cerebellar membranes has been further characterised, is reversible, and saturable, and exhibits high specificity for inositol polyphosphates. The IC50 for competition by unlabelled InsP6 is approximately 100nM, whereas inositol 1,3,4,5,6 pentakisphosphate (Ins(13456)P5), inositol 1,3,4,5 tetrakisphosphate (Ins(1345)P4), and inositol 1,4,5 trisphosphate (Ins(145)P3) bind with an affinity at least one order of magnitude lower. [3H]InsP6 binding is clearly distinct from previously characterised Ins(145)P3 (ref. 1, 2) and Ins(1345)P4 (ref. 3) binding, both in terms of pharmacology and brain distribution.

Nitric oxide in the rat gastrointestinal tract

This study concerns the nature of nitric oxide synthase (NOS) and the role of nitric oxide (NO) in the rat gastrointestinal tract. The major objectives were (i) to characterise NOS isoforms in the gastric glandular mucosa, (ii) to localise NOS isoforms in the rat gastric glandular mucosa, (iii) to investigate the role of NO in carbachol-stimulated gastric mucus secretion, (iv) to investigate the nature of NOS and small intestine. Immunoblotting was performed using polyclonal antisera raised against two peptides found in the rat brain NOS sequence and commercial monoclonal antibodies directed against neuronal and endothelial isoforms of NOS. A160kDa band was detected in brain and gastric mucosal samples with antibodies and antisera directed against neuronal NOS sequences, and a 140kDa band was detected in gastric mucosal samples using an anti-endothelial NOS antibody. An intense 160kDa neuronal NOS band was detected in a high-density fraction of gastric mucosal cells separated on a Percoll gradient. Detection of neuronal NOS by a carboxyl-terminal antiserum in samples of brain, but not of gastric mucosa, could be blocked by the peptide (20g/ml) against which the antibody was raised. After affinity purification, recognition of gastric mucosal NOS was blocked by peptide. Particulate neuronal NOS was found in the brain by immunoblotting while 94% of gastric mucosal enzyme was soluble. Gastric mucosal endothelial NOS was 95% particulate. 95% of NOS activity in the gastric mucosa was due to neuronal NOS. Paraformaldehyde- and acetone-fixed gastric mucosal sections were subject to immunocytochemistry using the above antibodies. Neuronal NOS was localised to the surface mucosal epithelial cells while endothelial NOS was associated with microvessels at the base of the mucosa and to larger vessels in the submucosa. Intragastric administration of carbachol or 16, 16-dimethyl prostaglandin E2 increased the thickness of the rat gastric mucus layer. The NOS inhibitor NG-nitro-L-arginine methyl ester dose-dependently, and selectively, prevented the stimulatory effect of carbachol. Ca2+-independent NOS activity in rat ileal, jejunal and colonic muscle was increased after LPS induction. Ca2+-dependent activity was not affected. Distribution of inducible NOS protein paralleled Ca2+ -independent activity. LPS treatment did not affect the content of neuronal NOS in colonic muscle.

Tetrahydrobiopterin metabolism in depression and senile dementia of Alzheimer type

Excretion of biopterin and the related pteridines neopterin and pterin was measured in urine samples from a group of 76 male and female unipolar and bipolar depressed outpatients receiving lithium therapy, and compared to 61 male and female control subjects. The ratio of neopterin to biopterin excreted (N/B) was significantly higher in the patients than the controls. The significant positive correlation between urinary neopterin and biopterin shown by the controls was absent in the patients, indicating disrupted biosynthesis of tetrahydrobiopterin.Urinary cortisol excretion in depressed patients was similar to controls, implying normal hypothalmus-pituitary-adrenal axis function in these patients, Serum folate was shown to correlate with urinary total biopterin excretion in female unipolar patients. Two groups of elderly females with senile dementia of Alzheimer type (SDAT) were examined for urinary pteridine excretion. In the first study of 10 patients, the N/B ratio was significantly higher than in 24 controls and the ratio B/B+ N significantly lower. A second study of 30 SDAT patients and 21 controls confirmed these findings. However, neopterin correlated with biopterin in both patients and controls, indicating that the alteration in tetrahydrobiopterin metabolism may be different to that shown in depression. Lithium had no effect in vivo or in vitro on Wistar rat brain or liver biosynthesis of tetrahydrobiopterin at a range of concentrations and duration of dosing period, showing that lithium was not responsible for the lowered biopterin excretion by depressed patients. No significant effects on tetrahydrobiopterin metabolism in the rat were shown by the tricyclic antidepressant imipramine, the anticonvulsant sodium valproate, the vitamin folic acid, the anticatecholaminergic agent amethylparatyrosine, the synthetic corticosteroid dexamethasone, or stimulation of natural cortisol by immobilisation stress. Scopolamine, an ant ichol inergic drug, lowered rat brain pterin which may relate to the tetrahydrobiopterin deficits shown in SDAT.

Tetrahydrobiopterin metabolism, neurotransmitters and behaviour in the rat

Various neurotoxins were investigated to assess their suitability for developing an animal model to study partial brain BH4 deficiency, neurotransmitters and behavioural alterations. Acute dosing with lead, diethylstilboestrol (DES), amphetamine and scopolamine produced no significant changes in rat brain BH4 metabolism though total biopterins in the liver were significantly reduced by lead and DES. Acute starvation of adult rats decreased brain biopterins. This loss of biopterins may be due to enhanced oxidative catabolism of the active cofactor caused by glutathione depletion. Dietary administration of a BH4 biosynthesis inhibitor, DAHP, consistently decreased brain total biopterins in weaner rats but did not alter the levels of DA, NA, 5-HT or metabolites. However the DAHP diet also induced a marked reduction in food intake. Rats subjected to an equivalent degree of food restriction without inhibitor showed significant but less severe reductions in brain biopterins and again no effect on transmitter levels. DAHP produced a significant decrease in locomotor activity and rearing. This could not be ascribed to reduction in food intake as animals subjected to just dietary restriction showed an increase in these activities. As gross brain levels of DA, NA and 5-HT were unaltered by DAHP the behavioural changes associated with the induced deficiency in brain total biopterins might not have been mediated through the action of these compounds. Although localised changes in neurotransmitter levels may have been obscured by gross analysis it is also possible that the behaviour changes were mediated by a role of BH4 not yet elucidated. Long-term administration of a high aluminium low calcium diet to mice produced no effect on gross brain total biopterins, catecholamines, serotonin or choline acetyltransferase activity though significant behavioural changes were observed.

The role of cannabinoid receptors in modulation of GABAergic neurotransmission in the rat medial entorhinal cortex in vitro

Type 1 cannabinoid receptors (CB1R) have a well established role in modulating GABAergic signalling with the central nervous system, and are thought to be the only type present at GABAergic presynaptic terminals. In the medial entorhinal cortex (mEC), some cortical layers show high levels of ongoing GABAergic signalling (namely layer II) while others show relatively low levels (layer V). Using whole-cell patch clamp techniques, I have, for the first time, demonstrated the presence of functional CB1R in both deep and superficial layers of the mEC. Furthermore, using a range of highly specific ligands for both CB1R and CB2R, I present strong pharmacological evidence for CB2Rs being present in both deep and superficial layers of the mEC in the adult rat brain. In brain slices taken at earlier points in CNS development (P8-12), I have shown that while both CB1R and CB2R specific ligands do modulate GABAergic signalling at early developmental stages, antagonists/ inverse agonists and full agonists have similar effects, and serve only to reduce GABAergic signalling. These data suggest that the full cannabinoid signalling mechanisms at this early stage in synaptogenesis are not yet in place. During these whole-cell studies, I have developed and refined a novel recording technique, using an amantidine derivative (IEM1460) which allows inhibitory postsynaptic currents to be recorded under conditions in which glutamate receptors are not blocked and network activity remains high. Finally I have shown that bath applied CB1 and CB2 receptor antagonists/ inverse agonists are capable of modulating kainic acid induced persistent oscillatory activity in mEC. Inverse agonists suppressed oscillatory activity in the superficial layers of the mEC while it was enhanced in the deeper layers. It seems likely that cannabinoid receptors modulate the inhibitory neuronal activity that underlies network oscillations.