Recurrent facial palsy: A CARE case report.

INTRODUCTION: Middle-ear adenoma is a rare benign endocrine tumor with slow progression, and can, in very rare cases, lead to recurrent peripheral facial palsy. CASE REPORT: A young man experienced three episodes of right peripheral facial palsy of incremental intensity, suggestive of barotrauma. CT and MRI found a tissue mass in the tympanic cavity, and biopsy diagnosed middle-ear adenoma. Electroneuromyography found 50% impairment of facial function. Closed right tympanoplasty with complete tumor resection enabled complete recovery of facial function within 1 month. DISCUSSION: Middle-ear adenoma is diagnosed on histology, as imaging on MRI can be non-specific, mimicking chronic otitis. Facial involvement is rare and is due to edematous compression of the vasa nervorum. Treatment is surgical, and follow-up should be prolonged. Palsy assessment on electroneuromyography indicates the urgency of treatment.

Bcl2, a transcriptional target of p38alpha, is critical for neuronal commitment of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF) cytokine. LIF starvation leads to cell commitment, and part of the ES-derived differentiated cells die by apoptosis together with caspase3-cleavage and p38alpha activation. Inhibition of p38 activity by chemical compounds (PD169316 and SB203580), along with LIF withdrawal, leads to different outcomes on cell apoptosis, giving the opportunity to study the influence of apoptosis on cell differentiation. By gene profiling studies on ES-derived differentiated cells treated or not with these inhibitors, we have characterized the common and specific set of genes modulated by each inhibitor. We have also identified key genes that might account for their different survival effects. In addition, we have demonstrated that some genes, similarly regulated by both inhibitors (upregulated as Bcl2, Id2, Cd24a or downregulated as Nodal), are bona fide p38alpha targets involved in neurogenesis and found a correlation with their expression profiles and the onset of neuronal differentiation triggered upon retinoic acid treatment. We also showed, in an embryoid body differentiation protocol, that overexpression of EGFP (enhanced green fluorescent protein)-BCL2 fusion protein and repression of p38alpha are essential to increase formation of TUJ1-positive neuronal cell networks along with an increase in Map2-expressing cells.

Unraveling substantia nigra sequential gene expression in a progressive MPTP-lesioned macaque model of Parkinson's disease.

Taking advantage of a progressive nonhuman primate model mimicking Parkinson's disease (PD) evolution, we monitored transcriptional fluctuations in the substantia nigra using Affymetrix microarrays in control (normal), saline-treated (normal), 6 days-treated (asymptomatic with 20% cell loss), 12 days-treated (asymptomatic with 40% cell loss) and 25 days-treated animals (fully parkinsonian with 85% cell loss). Two statistical methods were used to ascertain the regulation and real-time quantitative PCR was used to confirm their regulation. Surprisingly, the number of deregulated transcripts is limited at all time points and five clusters exhibiting different profiles were defined using a hierarchical clustering algorithm. Such profiles are likely to represent activation/deactivation of mechanisms of different nature. We briefly speculate about (i) the existence of yet unknown compensatory mechanisms is unraveled, (ii) the putative triggering of a developmental program in the mature brain in reaction to progressing degeneration and finally, (iii) the activation of mechanisms leading eventually to death in final stage. These data should help development of new therapeutic approaches either aimed at enhancing existing compensatory mechanisms or at protecting dopamine neurons.

The role of presenilin-1 in the gamma-secretase cleavage of the amyloid precursor protein of Alzheimer's disease.

Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells.

Molecular cloning, functional expression, pharmacological characterization and chromosomal localization of the human metabotropic glutamate receptor type 3.

Glutamic acid is the major excitatory amino acid of the central nervous system which interacts with two receptor families, the ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are coupled to G proteins and can be divided into three subgroups based on their sequence homology, signal transduction pathway and pharmacology. In this study, we describe the cloning of the cDNA encoding the human metabotropic glutamate receptor type 3 (HmGluR3). It was obtained by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to highly conserved sequences between rat mGluRs. The receptor shows 879 amino acids with 96% amino acid sequence identity with rat mGluR3. It is strongly expressed in fetal and adult whole brain, especially in caudate nucleus and corpus callosum. The gene was identified by fluorescence in situ hybridization on chromosome 7 band q22. Activation of the human mGluR3, permanently expressed in Baby Hamster Kidney (BHK) cells, by excitatory amino acid inhibits the forskolin-stimulated accumulation of intracellular cAMP. The rank order of potency is L-glutamic acid > or = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD) >> ibotenic acid > quisqualic acid. (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mM] is without effect on inhibition of forskolin-induced cAMP accumulation by L-glutamic acid.

The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line.

The human galanin receptor has been characterized pharmacologically from the Bowes melanoma cell line. Using porcine [125I]galanin as the radioligand, a single population of non-interacting high-affinity binding sites (KD = 0.05 +/- 0.01 nM; Bmax = 135 +/- 7 fmol/mg protein) was demonstrated. Human galanin peptide competitively inhibited the specific binding of [125I]galanin (IC50 = 0.35 +/- 0.13 nM) and decreased the forskolin-stimulated cAMP production (EC50 = 0.46 +/- 0.05 nM) with a maximal inhibition of 63 +/- 2% at 10(-7) M. Rat and porcine galanin peptides and the chimeric peptides M15, M35, M32, M40 and C7 also dose-dependently inhibited the forskolin-stimulated cAMP production, while the fragment porcine galanin-(3-29) and [D-Trp2]galanin were found to be inactive. The specific binding of [125I]galanin was decreased in a dose-dependent manner by GTP and the cAMP response was inhibited by the pertussis toxin, suggesting the activation of a G-protein dependent process. The Bowes cell line thus appears to be a relevant tool for the study of human galanin receptor.

Septide: an agonist for the NK1 receptor acting at a site distinct from substance P.

The hexapeptide [pGlu6,Pro9]substance P (SP)6-11, septide, has been shown to be an agonist as potent as SP in eliciting smooth muscle contraction in several in vitro preparations, while being a poor competitor of labeled SP binding. These results, as well as other pharmacological data, have suggested the existence of either a specific septide receptor or a septide site on the neurokinin (NK)1 receptor distinct from that for SP. We have used rat recombinant NK1 receptor expressed in COS-1 cells to address this issue. Both functional (agonist-induced inositol phosphate accumulation) and radioligand binding studies were conducted on transiently transfected cells. SP and septide elicited similar maximal increases (4-6-fold) in inositol phosphate levels in transfected cells, with EC50 values of 0.05 +/- 0.02 nM for SP and 5 +/- 2 nM for septide. No additivity of the maximal responses to the two agonists was observed, and neither agonist evoked any response in sham-transfected cells. RP 67580 was a competitive inhibitor of SP responses, with an inhibition constant (KB) of 13 +/- 2 nM, in agreement with displacement studies of [3H]SP binding to membranes and intact transfected cells (Ki values of 10 +/- 4 nM, and 1.16 +/- 0.06 nM, respectively). In comparison, septide responses were inhibited by RP 67580 in an uncompetitive fashion, with an apparent KB* value of 1.5 +/- 0.2 nM. Septide was a weak competitor of [3H]SP binding, with dissociation constants (Ki) of 2.9 +/- 0.6 microM and 3.7 +/- 0.9 microM for membranes and intact transfected cells, respectively. Similarly, septide at concentrations up to 10 microM did not affect [3H]RP 67580 binding. In conclusion, we have demonstrated that septide is a potent functional agonist of the NK1 receptor but it seems to act at a specific subsite different from that for SP. Although not ruling out the existence of selective septide receptors in some tissues, these results could explain some of the discrepancies with regard to the pharmacological properties of septide. Furthermore, a specific septide site on the NK1 receptor could represent an original pharmacological target.

Characterization of a human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG.

The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L-Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was approximately 1,000-fold less potent. The most potent peptide antagonist was trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N-methyl-N-(phe nylmethyl)-L- tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)-CP 96,345 > (+/-)-CP 96,345 > (+/-)-2-chlorobenzylquinuclidinone > (R,R)-CP 96,345 > RP 67580 > RP 68651. In [3H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.

Pharmacological characterization of RP 62203, a novel 5-hydroxytryptamine 5-HT2 receptor antagonist.

1. RP 62203 (2-[3-(4-(4-fluorophenyl)-piperazinyl)propyl]naphto[1,8- ca]isothiazole-1,1-dioxide) is a novel naphtosultam derivative which shows very high affinity for 5-HT2 receptors in the rat cerebral cortex (Ki = 50.0 pM). 2. RP 62203 is relatively selective for this sub-type of 5-hydroxytryptamine (5-HT) receptor, having lower affinity for the 5-HT1A receptor and very low affinity for the 5-HT, receptor. RP 62203 displayed low to moderate affinity for alpha 1-adrenoceptors, dopamine D2 receptors and histamine H1 receptors. 3. In vivo binding experiments demonstrated that oral administration of low doses of RP 62203 led to a long-lasting (greater than 6 h) occupation of cortical 5-HT2 receptors (ID50 = 0.39 mgkg-1). 4. In cortical slices from the neonatal rat, RP 62203 potently inhibited inositol phosphate formation evoked by 5-HT, with an IC50 of 7.76 nM. 5. The activity of neurones in the raphé and their responses to microiontophoretically applied 5-HT were studied with extracellular recording electrodes in the anaesthetized rat. RP 62203 potently and dose-dependently blocked excitations evoked by 5-HT when administered at doses of 0.5-4.0 mg kg-1, i.p. In contrast, neither 5-HT-evoked depressions nor glutamate-evoked excitations of raphé neuronal firing were blocked by RP 62203 at doses as high as 8.0 mg kg-1, i.p. 6. Head twitches induced by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) could be abolished by low doses of RP 62203 in mice (ED50 = 0.44 mg kg-1, p.o.) and in rats (ED50 = 1.54 p.o.). Similar results were obtained with mescaline and 5-hydroxytryptophan (5-HTP). 7. The potency of RP 62203 was compared with that of three other 5-HT2 receptor antagonists, ritanserin, ICI 169,369 and ICI 170,809. In all models, RP 62203 showed similar activity to ritanserin, whilst either ICI 169,369 or ICI 170,809 was several fold less active. 8. It is concluded that RP 62203 is a potent and selective antagonist at 5-HT2 receptors in the rodent central nervous system.

Partial purification and pharmacology of peripheral-type benzodiazepine receptors.

This report describes the results obtained with a new photoaffinity ligand for the "peripheral-type" benzodiazepine binding site (PBS), using a digitonin solubilized preparation from rat heart or adrenals. The specific binding activity of the solubilized adrenal preparation is higher than 50 pmol/mg protein, with binding properties and pharmacological specificity identical to the membrane bound PBS. The apparent molecular weight of the solubilized PBS, determined by gel filtration is 215 KDa. The photoaffinity ligand (PK 14105) is a nitrophenyl derivative of PK 11195, which attaches covalently and specifically to all the PBS when cardiac membranes are irradiated with this compound under ultraviolet light. After photolabelling with [3H]PK 14105 and solubilization in SDS of heart or adrenal membranes, gel electrophoresis indicates the existence of a single protein band whose molecular weight (18 KDa) is unaltered by incubation with sulphydryl-reducing or protein cross-linking agents. This molecule seems to be a low molecular weight, acidic protein. Diethylpyrocarbonate decreases partially (60%) the binding of [3H]PK 11195 without affecting [3H] RO5-4864 binding, which implies a vital histidine residue in the binding domain of [3H]-PK 11195. Treatment with phospholipase A2 or mellitin, a stimulant of endogenous PLA2, led to a selective loss of [3H] RO5-4864 binding with no change in the binding of [3H]PK 11195. Such differences between a benzodiazepine ligand and an isoquinoline ligand suggest that these compounds may induce, on binding, different conformational changes in the PBS, which is compatible with the hypothesis that RO5-4864 and PK 11195 may be an agonist and an antagonist respectively at the PBS.

Photoaffinity labeling of peripheral-type benzodiazepine-binding sites.

The use of a novel photoaffinity label for the peripheral-type benzodiazepine-binding site is described. This compound, PK 14105, has high affinity (4 nM) and selectivity for cardiac benzodiazepine-binding sites. Under ultraviolet light, PK 14105 couples covalently to an 18,000-Da membrane protein which apparently corresponds to the (or a part of the) cardiac benzodiazepine-binding site. Since covalent attachment of PK 14105 totally precludes the binding of other ligands to this binding site, it is suggested that, during ultraviolet irradiation, this compound inserts covalently into the binding domain of the peripheral-type benzodiazepine-binding site.

Dihydropyridine and peripheral type benzodiazepine binding sites: subcellular distribution and molecular size determination.

Electrophysiological and pharmacological studies have shown that peripheral-type benzodiazepine receptors modulate voltage-sensitive calcium channels in the heart. We have compared these binding sites with binding sites for [3H]dihydropyridines, which are believed to label such channels. Although no direct or allosteric interaction could be demonstrated between the two sites, their subcellular distribution--sarcolemma and ryanodine-sensitive sarcoplasmic reticulum--was parallel. Size determination of the two sites suggests that the receptors for these two classes of compounds are separate molecules packaged in the same membrane compartment.

Characterization of solubilized "peripheral type" benzodiazepine binding sites from rat adrenals by using [3H]PK 11195, an isoquinoline carboxamide derivative.

"Peripheral type" benzodiazepine binding sites have been solubilized with digitonin. Binding site density for the solubilized material is increased 1.7 times compared to membranes. A decrease in the affinity for [3H]-PK 11195 (a new ligand for the peripheral type benzodiazepine binding sites) was also observed. Pharmacological specificity of displacing agents was conserved during solubilization. The apparent molecular weight determined by gel filtration was 215,000 +/- 20,000. The high Bmax value of the solubilized preparation (greater than 50 pmole/mg protein) makes it advantageous as the starting point for a purification procedure.