Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs

Nearly 7000 Arabidopsis thaliana -expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.     

Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.     

A method facilitating the demonstration of synergism in the tissue-responses to mixed peptide stimuli

A methodology for the pharmacodynamical assay of mixtures of 2 spasmogenic peptides is described, illustrated and discussed, with reference to a method intended for the chemists (P. Job, 1927). This allows a quick appreciation of the existence of synergism, of its own order of magnitude and of that of the doses for which it is significant. Also given is a fast way of curve-fitting the dose response to individual peptides, according to Clark's approximation.     

Spot position of rat liver ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel

Summary Separation of the proteins from rat liver 40S and 60S ribosomal subunits and polysomes was done in four different two-dimensional polyacrylamide gel electrophoresis systems. The first dimension was run at acidic or basic pH, the second dimension either with sodium dodecyl sulphate or at acidic pH in 18% acrylamide. The position of each individual protein of both subunits and polysomes was determined in each system. This identification resulted from a new method avoiding any previous purification of individual proteins. The new proposed uniform nomenclature for mammalian ribosomal proteins (McConkey et al. in press) was used for numbering the proteins in the four systems.     

Beta-VLDL increases endothelial cell plasma membrane cholesterol

In this study, the distribution of free cholesterol in cholesterol-loaded endothelial cells was examined. For these studies, cell fractionation methods were used to assess marker enzyme activity and cholesterol distribution. Treatment of rabbit aortic endothelial cells for 3 days with 50 micrograms/ml of beta-very low density lipoprotein (beta-VLDL) or malondialdehyde-low density lipoprotein (MDA-LDL) but not LDL caused a 50-100% increase in total cell unesterified cholesterol. The accumulation of free rather than esterified cholesterol in endothelial cells may be due to the ratio of hydrolysis to esterification, which we have shown in this study to be 10-fold higher in endothelial cells than in smooth muscle cells. This free cholesterol is found in the fractions enriched in plasma membrane markers and, to a lesser extent, in the Golgi-enriched fractions. The amount of cholesterol per mg of protein was increased approximately 50% in these fractions from cells treated for 3 days with 50 micrograms/ml of beta-VLDL. These increases in cholesterol content were reversible upon incubation of cells for 3 days in medium containing 15% fetal bovine serum. Alterations in several membrane functions were also observed in cholesterol-loaded cells. The activity of alkaline phosphatase, an enzyme marker for plasma membranes, was decreased by 25% and an alteration in membrane-associated microfilaments was seen with phalloidin staining. This morphological change in microfilaments was reflected in a decrease in filament ends as shown by cytochalasin binding and occurred without a change in total actin or vinculin. These microfilament changes were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular Events Induced by γ-Hydroxybutyrate in Striatum: A Microdialysis Study

The modification of dopamine release and accumulation induced by gamma-hydroxybutyrate (GHB) was studied using both striatal slices and in vivo microdialysis of caudate-putamen. GHB inhibited dopamine release for approximately 5-10 min in vitro, and this was associated with an accumulation of dopamine in the tissue. Subsequently, there was an increase in dopamine release. In the microdialysis experiments, low doses of GHB inhibited dopamine release, whereas higher doses strongly increased release; the initial decrease seen in slices could not be detected in vivo. Thus, GHB had a biphasic effect on the release of dopamine: An initial decrease in the release of transmitter was followed by an increase. A time-dependent biphasic effect was observed when GHB was added to brain slices, and a dose-dependent biphasic effect was seen in dialysate after systemic administration of GHB. Naloxone blocked GHB-induced dopamine accumulation and release both in vitro and in vivo. GHB also increased the release of opioid-like substances in the striatum. A specific antagonist of GHB receptors completely blocked both the dopamine response and the release of opioid-like substances. These data suggest that GHB increases dopamine release via specific receptors that may modulate the activity of opioid interneurons.