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Biomedical subjects

V E Shashoua

Publications and source records attributed to V E Shashoua.

At least 19 recordsLinked to original sources
PubMed2001

CMX-8933, a peptide fragment of the glycoprotein ependymin, promotes activation of AP-1 transcription factor in mouse neuroblastoma and rat cortical cell cultures.

An 8-amino acid peptide fragment (CMX-8933) of Ependymin, a glycoprotein component of the extracellular fluid and cerebrospinal fluid of goldfish brain, was synthesized and tested for its capacity to activate AP-1 transcription factor in cell cultures. Dose-response and time-course studies of AP-1's binding to DNA were carried out in neuroblastoma (NB2a/dl) and primary rat brain cortical cultures using an electrophoretic mobility shift assay (EMSA). A 13-14-fold increase in AP-1's DNA binding was obtained when NB2a cells were incubated for 4 h with 6-10 microg/ml CMX-8933. Primary rat brain cortical cultures were much more sensitive to the effects of CMX-8933 than transformed (NB2a) cultures; here a 26.7+/-5.2-fold increase in binding was observed following a 3-h treatment with as little as 10 ng/ml peptide. These findings are consistent with an activation of this transcription factor, a characteristic that has been previously correlated with functional aspects of full-sized neurotrophic factors (nerve growth factor and brain-derived nerve growth factor) in neuronal differentiation and regeneration. Such data suggest a role for Ependymin in transcriptional control.

Animals↗
PubMed2001

Fatty acid derivatives of clozapine: prolonged antidopaminergic activity of docosahexaenoylclozapine in the rat.

Stable amides of clozapine derived from fatty acids prominent in cerebral tissue might enhance the central activity of clozapine and reduce its exposure to peripheral tissues. Such derivatives might enhance the safety of this unique drug, which is the only agent with securely established superior antipsychotic effectiveness, but with a risk of potentially lethal systemic toxicity. Amide derivatives of clozapine were prepared from structurally varied fatty acid chlorides and evaluated for ability to inhibit behavioral arousal in rat induced by dopamine agonist apomorphine and to induce catalepsy. Their duration-of-action and potency were compared to free clozapine, and concentrations of clozapine were assayed in brain and blood. Selected agents were also evaluated for affinity at dopamine receptors and other potential drug-target sites. Clozapine-N-amides of linoleic, myristic, oleic, and palmitic acids had moderate initial central depressant activity but by 6 h, failed to inhibit arousal induced by apomorphine. However, the docosahexaenoic acid (DHA) derivative was orally bioavailable, 10-times more potent (ED(50) 5.0 micromol/kg) than clozapine itself, and very long-acting (>/= 24 h) against apomorphine, and did not induce catalepsy. DHA itself was inactive behaviorally. Clozapine showed expected dopamine receptor affinities, but DHA-clozapine was inactive at these and other potential target sites. After systemic administration of DHA-clozapine, serum levels of free clozapine were very low, and brain concentrations somewhat lower than after administering clozapine. DHA-clozapine is a long-acting central depressant with powerful and prolonged antidopaminergic activity after oral administration or injection without inducing catalepsy, and it markedly reduced peripheral exposure to free clozapine. It lacked the receptor-affinities shown by clozapine, suggesting that DHA-clozapine may be a precursor of free, pharmacologically active clozapine. Such agents may represent potential antipsychotic drugs with improved central/peripheral distribution, and possibly enhanced safety.

Amides↗
PubMed1996

Genes encoding giant danio and golden shiner ependymin.

Ependymin (EPN) is a brain glycoprotein that functions as a neurotrophic factor in optic nerve regeneration and long-term memory consolidation in goldfish. To date, true epn genes have been characterized in one order of teleost fish, Cypriniformes. In the study presented here, polymerase chain reactions were used to analyze the complete epn genes, gd (1480 bp), and sh (2071 bp), from Cypriniformes giant danio and shiner, respectively. Southern hybridizations demonstrated the existence of one copy of each gene per corresponding haploid genome. Each gene was found to contain six exons and five introns. Gene gd encodes a predicted 218-amino acid (aa) protein GD 93 percent conserved to goldfish EPN, while sh encodes a predicted 214-aa protein SH 91 percent homologous to goldfish. Evidence is presented classifying proteins previously termed "EPNs" into two major categories: true EPNs and non-EPN cerebrospinal fluid glycoproteins. Proteins GD and SH contain all the hallmark, features of true EPNs.

Amino Acid Sequence↗
PubMed1996

N-docosahexaenoyl, 3 hydroxytyramine: a dopaminergic compound that penetrates the blood-brain barrier and suppresses appetite.

Fatty acids with varying chain lengths (2-22 carbon atoms long) and degrees of unsaturation (0-6 double bonds) were used to synthesize dopaminergic compounds for a study of the carrier mediated transport of dopamine (DA) to the brain. The most active carrier was the all cis C22:6 fatty acid [docosahexaenoic acid, (DHA)]which increased DA uptake through the blood-brain barrier by greater than 7.5 fold. The DHA-DA compound, NMI 8739, depressed the general locomotor activity of mice in a dose dependent manner. It also suppressed the appetite of Balb c mice and Charles River rats by 50% and 95% respectively at a dose of 10 mg/kg. Daily administration of NMI-8739 for a three week period did not induce tolerance. These results demonstrate DHA's potential for the carrier mediated transport of small molecules to the brain.

Animals↗
PubMed1994

Cloning and sequencing the genes encoding goldfish and carp ependymin.

Ependymins (EPNs) are brain glycoproteins thought to function in optic nerve regeneration and long-term memory consolidation. To date, epn genes have been characterized in two orders of teleost fish. In this study, polymerase chain reactions (PCR) were used to amplify the complete 1.6-kb epn genes, gf-I and cc-I, from genomic DNA of Cypriniformes, goldfish and carp, respectively. Amplified bands were cloned and sequenced. Each gene consists of six exons and five introns. The exon portion of gf-I encodes a predicted 215-amino-acid (aa) protein previously characterized as GF-I, while cc-I encodes a predicted 215-aa protein 95% homologous to GF-I.

Amino Acid Sequence↗
PubMed1992

Dibutyryl cyclic AMP stimulates expression of ependymin mRNA and the synthesis and release of the protein into the culture medium by neuroblastoma cells (NB2a/d1).

Northern blot, immunoprecipitation, and gel electrophoretic data demonstrate that the mouse neuroblastoma NB2a/d1 cells express ependymin mRNA and synthesize and release into the culture medium a protein with immunoreactivity and electrophoretic mobility properties identical to ependymin. This is a brain extracellular glycoprotein that has been implicated in the consolidation process of memory formation and neuronal regeneration. In labeling experiments with 35S-methionine, dibutyrylcyclic3',5'-adenosine-monophosphate (dbcAMP) was found to stimulate the expression of ependymin mRNA and the enhanced synthesis and release of ependymin into the culture medium at the same time that dbcAMP stimulation of neurite outgrowth takes place. These results are consistent with the proposed role of the protein in the mechanism of neuronal regeneration and synaptogenesis. The data indicate that the NB2a/d1 cell line is a good model system for studies of the functional properties of ependymin.

Amino Acid Sequence↗
PubMed1991

Ependymin, a brain extracellular glycoprotein, and CNS plasticity.

Ependymin, a glycoprotein of the brain ECF, has been implicated in the neurochemistry of memory and neuronal regeneration. Three behavioral experiments (swimming with a float, avoidance conditioning, and classical conditioning) in the goldfish and one in the mouse (T-maze learning) indicate that ependymin has a role in the synaptic changes that take place in the consolidation step of memory formation and the activity-dependent phase of sharpening of goldfish retinotectal connections during neuronal regeneration. The ECF concentration of the protein was found to decrease after the goldfish learned to associate a light stimulus (CS) with the subsequent arrival of a shock (US): paired CS-US gave changes whereas an unpaired presentation of CS-US gave no changes relative to the unstimulated controls. Ependymin is present in ECF as a mixture of three disulfide-linked dimers of two acidic (alpha and beta) polypeptide chains (37 kDa and 31 kDa). Upon removal of its N-linked glycan fragment by N-glycosidase F, the beta chain yields gamma-ependymin (26 kDa). Determinations of the amino acid sequence of gamma-ependymin indicate that it is a unique protein with no long sequence homologies to any known polypeptide. There are, however, small segments (5-7 amino acids long) with homologies to fibronectin, laminin, and tubulin. Ependymin has the capacity to polymerize into FIP (after activation by phosphorylation) in response to events that deplete ECF calcium. FIP is insoluble in 2% SDS in 6 M urea, 10 mM Ca2+Ac2, 100% acetic acid, chloroform/methanol (2/1), saturated KCNS, and even 100% trifluoroacetic acid. FIP was found to be present in goldfish brain and to be formed as a labeled product in vivo. Ependymin's FIP-forming property was used to propose a molecular hypothesis for generating synaptic changes in response to local extracellular depletions of calcium at sites of "associating inputs." The model assumes that, following NMDA receptor stimulation, the translocated PKC that is generated activates extracellular ependymin by converting it to its phosphorylated form using presynaptically released ATP. The hypothesis was tested in studies of LTP of rat hippocampal slices at CA1. After LTP, new sites that stained with antisera to ependymin, visible at 100x, were obtained in its potentiated radiatum in the CA1 region but not in the unpotentiated CA3. Electron microscopic studies showed that the horseradish peroxidase reaction product obtained was localized at synaptic clefts and postsynaptic regions. The results suggest that FIP may be formed at extracellular and postsynaptic loci where multiple associating inputs interact at CA1.

Animals↗
PubMed1990

Evidence for the in vivo polymerization of ependymin: a brain extracellular glycoprotein.

Ependymin, a glycoprotein of the brain extracellular fluid, has been implicated in synaptic changes associated with the consolidation process of long-term memory formation and the activity-dependent sharpening of connections of regenerating optic nerve. In vitro experiments have demonstrated that ependymin has the capacity to form fibrous insoluble polymers (FIP) when the solvent Ca2+ concentration is reduced by the addition of EGTA. Such products, once formed, do not dissolve in 2% sodium dodecyl sulfate (SDS) in 5 M urea. This property was used to develop a method for isolating brain FIP. A reproducible quantity of FIP was found in goldfish and mouse brain. This was highly concentrated in the synaptosomal fraction and had identical immunoreactivity properties to FIP obtained by the polymerization of pure ependymin in vitro as well as a cross-reactivity to other protein components of the extracellular matrix such as fibronectin and laminin. Labeling studies with [35S]methionine showed that labeled FIP aggregates are synthesized in vivo and become associated with the synaptosomal fraction. A comparison of the amino acid sequence of ependymin with those for proteins of the extracellular matrix indicated that common sequences 5-6 amino acids long exist in the molecules. These homologies may explain why antibodies to fibronectin, laminin and tubulin can recognize the FIP prepared from pure ependymin. These results suggest that ependymin can polymerize in vivo to form FIP aggregates which have similar immunoreactivity properties to major components of the brain extracellular matrix.

Amino Acid Sequence↗
PubMed1990

Protein synthesis as a function of depth in slices of rat hippocampus.

Physiologically viable slices of rat hippocampus were incubated in radiolabeled valine, then cut into 20 microns serial sections to evaluate the profile of protein synthesis through the depth of the slice. Maximum radiolabel incorporation was observed near the center of the slice, while at the upper (gas interface) and lower (liquid interface) surfaces radiolabel incorporation per section was reduced by about 30% and 90%, respectively. The results suggest that in properly slices damage due to slicing may be less important to cell viability than are limits on oxygen diffusion into the tissue.

Animals↗
PubMed1990

Synthesis, brain uptake, and pharmacological properties of a glyceryl lipid containing GABA and the GABA-T inhibitor gamma-vinyl-GABA.

1-O-Linolenoyl-2-O-(4-aminobutyryl)-3-O-(4-vinyl-4-aminobutyryl)glycerol (LGV) was synthesized as an example of a prodrug which readily penetrates the blood-brain barrier (brain penetration index 97% +/- 15%) and releases two active substances in the central nervous system (CNS): GABA (4-aminobutanoic acid) and the GABA transaminase inhibitor (GABA-T) of GABA breakdown. In vitro studies showed that the compound can inhibit GABA-T after hydrolysis by CNS esterases and that it enhanced GABAergic inhibition when applied to rat hippocampus slices. In vivo studies indicate that LGV depresses the spontaneous locomotor activity of mice. Its activity on a molar basis was some 300 times greater than that of gamma-vinyl-GABA.

4-Aminobutyrate Transaminase↗
PubMed1989

Classical conditioning leads to changes in extracellular concentrations of ependymin in goldfish brain.

ELISA measurements showed that brain extracellular fluid (ECF) levels of ependymin decreased for animals that learned to associate a paired presentation of a light stimulus (CS) with the onset of an electric shock (US), whereas no changes were obtained for control goldfish that received the same number of stimuli delivered in a random unpaired order. Studies of the time course of the changes showed an immediate decrease (19%) after training followed by an increase (20%) above baseline by 5 h and a final return to baseline by 25 h. These data extend the findings of previous experiments, which demonstrated a role for ependymin in two training procedures that involved motor learning, to classical conditioning where no motor learning occurs. Thus it appears that ependymin may have a functional role in molecular mechanisms of learning and memory in general.

Animals↗
PubMed1988

Antibodies to ependymin block the sharpening of the regenerating retinotectal projection in goldfish.

The regenerating optic nerve of goldfish first reestablishes a rough retinotopic map on the tectum, then goes through an activity dependent refinement that appears to involve the elimination of inappropriate branches from early regenerated arbors. Retinotopically appropriate branches and synapses may be stabilized because the normally correlated firing of neighboring ganglion cells could cause summation of their postsynaptic responses, making them more effective. Thus, refinement of the map may be similar in several ways to associative learning. In this study, we therefore tested whether ependymin, a major protein component of the extracellular fluid that has been implicated in synaptic changes thought to be associated with learning a simple task in goldfish, may also be involved in refinement of the retinotopic map. Goldfish that had undergone unilateral optic nerve crush received intraventricular infusion of antiependymin IgG or of control IgG's beginning at 21 days postcrush. Tectal recordings from these fish at 39-56 days postcrush showed that the projection had failed to sharpen, much as in the fish with activity blocked or synchronized; the average size of the multiunit receptive fields was 31 degrees vs 11 degrees normally. The field potentials elicited from these tecta by optic nerve shock were not significantly smaller than in controls, suggesting normal levels of synaptogenesis. Control projections, identically treated but infused with either unrelated IgG or Ringer's alone regenerated normally, giving multiunit receptive fields of 12 degrees. Intact (non-regenerating) projections of the experimental fish were not rendered abnormal by the IgG treatment. Histology showed the retinas and tecta of the infused fish to be normal in appearance. The results show a specific block of sharpening by antiependymin IgG. The ependymal glia of the tectum stain positively for ependymin in normal fish, particularly the cell bodies in the ependymal layer. The tectum, particularly the ependymal layer, stains more intensely during regeneration, which appears to trigger increased synthesis of ependymins in the ependymal glia. This increase and the block of sharpening by specific antibodies to ependymin suggest a possible role for ependymin in activity dependent synaptic stabilization, possibly through its polymerization when calcium is focally depleted at active synapses.

Action Potentials↗
PubMed1988

Monomeric and polymeric forms of ependymin: a brain extracellular glycoprotein implicated in memory consolidation processes.

Ependymin, a brain extracellular glycoprotein that appears to be implicated in neural circuit modifications associated with the process of memory consolidation, can rapidly polymerize into fibrous aggregates when the Ca2+ concentration in solution is reduced by the addition of EGTA or by dialysis. Such aggregates, once formed, could not be redissolved in boiling 1% SDS in 6 M urea, acetic acid, saturated aqueous potassium thiocyanate, and trifluoroacetic acid. They were, however, soluble in formic acid. Investigations of the immunological properties of ependymin indicated that various monomers, oligomers and polymers of the molecule with differing carbohydrate contents can be obtained. The polymerization properties of the ependymins may play an important role in their functions in memory consolidation mechanisms.

Animals↗
PubMed1988

Uptake in brain and neurophysiological activity of two lipid esters of gamma-aminobutyric acid.

Two lipid esters of gamma-aminobutyric acid (GABA), 1-linolenoyl-2,3-bis(4-aminobutyryl)propane-1,2,3-triol and 1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol, were found to have brain uptake indices of greater than 30% using the single-pass carotid artery injection technique. Both compounds produced dose-dependent inhibition of the evoked population spike in slices of rat hippocampus maintained in vitro. This effect was blocked reversibly by picrotoxin. The magnitude of the inhibition produced by the lipid esters of GABA was comparable to that of similar doses of GABA, but for both compounds the duration of the effect was at least 10 times longer than that produced by GABA. These data are consistent with the idea that the lipid esters of GABA can effectively penetrate the blood-brain barrier and act as prodrugs for the delivery of GABA to the central nervous system.

Action Potentials↗
PubMed1988

Novel GABA agonists depress the reward effect of lateral hypothalamic stimulation in rats.

Rats were given systemic injections of one of a series of novel GABA compounds which can penetrate the blood-brain barrier to release GABA into the brain. They were then tested on lateral hypothalamic self-stimulation behavior using a rate-frequency paradigm to discriminate effects on reward from those on motor/performance. Both reward and, to a lesser extent, motor/performance impairments were found with all GABA compounds. In more extensive testing with one compound, LG2, no differences in the effects of three salts (acetate, ascorbate, and tartarate) were found except that the tartarate salt effects decayed more rapidly.

Animals↗
PubMed1987

gamma-Aminobutyric acid esters. 3. Synthesis, brain uptake, and pharmacological properties of C-18 glyceryl lipid esters of GABA with varying degree of unsaturation.

A series of 14C-labeled and unlabeled di-gamma-aminobutyric acid esters of glyceryl lipids having zero to three double bonds (stearoyl, oleoyl, linoleoyl, and linolenoyl) were synthesized. Measurements of the octanol/water partition coefficients of the compounds showed an increase with decreasing number of double bonds (i.e., from linolenoyl to stearoyl). The brain-uptake index went up from 31.5 (linolenoyl) to 45.1 (stearoyl) and similarly the brain-penetration index went up from 15 (linolenoyl) to 28 (stearoyl). Intraperitoneal injections of these di-GABA lipid esters produced a substantial inhibition of the general motor activity in mice at a dose of 30 mg/kg; the most active molecules were those containing two and three double bonds, i.e., the linolenoyl and linolenoyl derivatives. This is in reverse order to that predicted by brain-uptake and lipid-solubility properties, suggesting that the structure of the fatty acid side chain may be an additional factor in influencing biological activity.

Animals↗
PubMed1986

Demonstration of glucuronic acid on brain glycoproteins which react with HNK-1 antibody.

Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.

Animals↗