The amyloid precursor protein of Alzheimer's disease is released by human platelets.

Western blots of normal human platelets, employing a monoclonal antibody raised against the full-length amyloid precursor protein of Alzheimer's disease (APP695), revealed major bands of 100-110 and 120-130 kDa in both cytosolic, membrane, and released fractions. These species were similar in size to forms seen in brain preparations and in plasma. There was no difference in Western blots of platelet preparations from Alzheimer patients compared with controls. Purified platelet amyloid precursor proteins were sequenced and shown to be amino terminally homogeneous. Immunohistochemistry localized the antigen to the platelet and megakaryocyte and demonstrated weak immunostaining of some lymphocytes. Immunoprecipitation of material released from platelets demonstrated that sedimentable full-length APP with the carboxyl-terminal epitope, and soluble APP lacking the carboxyl-terminal epitope, may exist in the circulation. Western blots and carboxyl-terminal and amino-terminal APP radioimmunoassay of material released by platelets in response to stimulation revealed that platelets release APP during degranulation. The function of platelet APP is yet to be determined, but the present studies suggest a role in regulation of the coagulation cascade or in platelet aggregation.

Improved quantification of in vivo 1H NMR spectra by optimization of signal acquisition and processing and by incorporation of prior knowledge into the spectral fitting.

Quantification of localized in vivo brain 1H spectra is in general very difficult due to excessive spectral overlap. In addition, intensity distortions may result from the effects of the NMR pulse sequence on the spins. This paper describes an approach to solving these problems. It comprises optimization of the pulse sequence; correction of the experimental lineshape; determination of intensity distortions, of relative line positions, and of linewidths using model solutions; and incorporation of the thus obtained prior knowledge into a nonlinear least-squares spectral fitting procedure. This approach resulted in greatly improved accuracy, precision, and reliability of the quantitation of our in vivo spectra of rat brain, and enabled us to estimate absolute metabolite concentrations.

Intermembrane transfer and antioxidant action of alpha-tocopherol in liposomes.

Intermembrane transfer and exchange of tocopherol are not well understood. To study this we tested the ability of alpha-tocopherol containing unilamellar donor liposomes to inhibit the accumulation of lipid peroxidation products in acceptor liposomes. With molar ratios of alpha-tocopherol:phospholipids from 1:100 to 1:1000 in donor liposomes prepared by sonication of lipid dispersions, alpha-tocopherol was incorporated into both monolayers and was homogenously distributed in monomeric form without forming clusters in the liposomes. Concentrations of alpha-tocopherol which completely prevented the peroxidation of lipids were chosen for donor liposomes. Hence inhibition of lipid peroxidation in mixtures of donor and acceptor liposomes was determined by the antioxidant effect of alpha-tocopherol in acceptor liposomes which resulted from intermembrane transfer and exchange of alpha-tocopherol. Evidence was obtained that this was not due to fusion of donor with acceptor liposomes. The efficiency of the "intermembrane" antioxidant action of tocopherol was more pronounced when donor liposomes contained unsaturated phospholipids, indicating that the presence of unsaturated fatty acids in the outer monolayer phospholipids facilitates intermembrane tocopherol exchange.

Biochemical assay of Alzheimer's disease--associated protein(s) in human brain tissue. A clinical study.

The concentration of Alzheimer's disease-associated protein (ADAP) was measured in postmortem brain tissue samples of temporal or frontal cortex from 111 human brains using a sandwich immunoassay. Alzheimer's disease-associated protein has three major ALZ-50-reactive subunits, including A-68. This assay utilizes ALZ-50 and a rabbit antibody raised against a highly ADAP-enriched brain protein fraction. The frequently observed cross-reactivity of ALZ-50 with normal brain components in direct immunoassays is minimized by this configuration. There were 27 normal controls, 28 neurologic disease controls, and 56 Alzheimer's disease cases. The normal control and neurologic disease control cases had essentially no detectable level of ADAP, while ADAP was clearly detected in 85.7% of the Alzheimer's disease cases. Clinical dementia, neuritic plaques, and old age per se are not correlated with increased ADAP levels. This biochemical assay of ADAP may prove to be helpful as an adjunct in the clinicopathologic diagnosis of Alzheimer's disease.

A major direct GABAergic pathway from zona incerta to neocortex.

Retrograde fluorescent tracers were used to demonstrate a previously unknown but sizable direct gamma-aminobutyric acid (GABA)-containing neuronal pathway from the zona incerta to the neocortex in rats. This incertocortical pathway was found to project bilaterally to the entire neocortex and exhibited a rough corticotopic organization. Many of the zona incerta neurons projecting to the parietal and occipital cortices could also be immunohistochemically stained with antibodies to glutamic acid decarboxylase and GABA. Few of these neurons were immunoreactive to tyrosine hydroxylase antibodies, which identify dopamine-containing neurons. Injections in the frontal and entorhinal cortices labeled many neurons near or within the dopaminergic A13 subdivision of the zona incerta. In addition, the incertocortical system was found to be significantly larger during early postnatal (2 to 3 weeks) development. The projection pattern of this newly discovered pathway resembles that of the monoaminergic and cholinergic systems, arising from the brainstem and forebrain, suggesting possible similarities of function.

Isolation and properties of the alpha-latrotoxin receptor.

The receptor protein of alpha-latrotoxin (alpha LTx, a neurotoxin with 'pure' presynaptic action isolated from black widow spider venom), was solubilized by Triton X-100 from bovine brain membranes and purified by affinity chromatography on alpha LTx-Sepharose. The purified receptor preparation contained four major polypeptides of molecular masses 200 (alpha), 160 (alpha'), 79 (beta) and 43 (gamma) kd according to SDS electrophoresis with molecular ratio alpha 1 alpha' 1 beta 2 gamma 2. The alpha- and alpha'-subunits are glycoproteins binding to wheat germ lectin and can be separated under non-denaturing conditions by anion exchange chromatography. Purified to homogeneity, both of them, though differing in the carbohydrate composition, retain the alpha LTx-binding activity and give closely related peptide maps. Anti-alpha antibodies recognize the alpha'-subunit as well. These results suggest that alpha LTx receptor is present in purified preparations in two very close forms containing the alpha- or alpha'-subunit. Beta and gamma proteins do not specifically bind alpha LTx and their physiological role is unclear. They form a complex with solubilized alpha- and alpha'-subunits independently of alpha LTx presence. The receptor proteins were purified to homogeneity by high performance gel filtration in the presence of SDS, their amino acid composition was determined.

Quantitative immunohistochemistry of synaptophysin in human neocortex: an alternative method to estimate density of presynaptic terminals in paraffin sections.

Currently available specific synaptic markers have made it possible to estimate the synaptic density by immunochemical techniques. In the present study we labeled the neocortical presynaptic terminals in histological sections of human autopsy tissue with a monoclonal antibody against synaptophysin. The characteristic granular neuropil reaction was quantified by measuring the average optical density (OD) in the different layers of the parietal cortex with the aid of image analysis equipment. The raw neuropil OD was corrected by subtracting the OD of the white matter in the same section. Our study showed that consistent microdensitometric results can be obtained on 5-microns paraffin sections from specimens with less than 8 hr of post-mortem time before fixation, incubated with 5 micrograms/ml of anti-synaptophysin. The corrected OD measurements were slightly larger in neocortical layers II, III, and V than in layers I, IV, and VI, but the differences were not statistically significant. In area 17, layer IV was denser than the others. We conclude that with certain precautions this method can be used to measure relative amounts of synaptophysin-like immunoreactivity and to infer the density of presynaptic boutons in human situations and in animal models.

Separation of two beta gamma subunit complexes of brain GTP-binding proteins composed of distinct gamma subunits.

The gamma subunits of GTP-binding proteins are always associated with beta subunits under physiological conditions, and at least two gamma subunits exist in the brain. We report here that brain beta gamma subunit complexes composed of distinct gamma subunits can be separated by anion exchange chromatography under nondenaturing conditions. One beta gamma complex was composed of a 36-kDa beta subunit and a 6-kDa gamma subunit and the other was composed of 36/35-kDa beta subunits and 4.5-kDa gamma subunit. The 6-kDa gamma subunit was phosphorylated by protein kinase C but the 4.5-kDa gamma subunit was not.

P29: a novel tyrosine-phosphorylated membrane protein present in small clear vesicles of neurons and endocrine cells.

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.

Improved method of analysis for aluminum in brain tissue.

To improve the accuracy and precision of the assay of aluminum in brain tissue, we modified for application to brain samples from rats and humans the wet-digestion method of Trapp et al. (Biol Psychiatry 1978; 13:709-18), established the contribution of contamination, and examined the effect of precipitation of nonoxidizable fatty residues on the analysis. Specifications of the modified assay are a detection limit of 5 ng of aluminum per gram wet weight of brain tissue, a within-day CV of 4.8% (24.3 microgram/L; n = 10), and a day-to-day CV of 5.5% (27.8 micrograms/L; n = 5). Contamination, a systematic error in the analysis of aluminum, was established to be 13 ng (SD = 7.9 ng; n = 8) per tube. The presence of indestructible fatty residues did not affect the accuracy of the method. Application of the method to brain hemispheres of nonexposed rats revealed an aluminum content of 0.041 mg/kg wet weight of tissue (SD = 0.032 mg/kg; n = 8). The aluminum content in human cortex samples, consisting of gray and white matter, ranged from 0.14 to 0.22 mg/kg. Modification of the wet-digestion method resulted in a reliable, simple sample pretreatment before analysis for aluminum in brain tissue. The extent of the aluminum contamination must be controlled by including appropriate blanks.

Molecular mechanisms of homologous desensitization and internalization of muscarinic receptors in primary cultures of neonatal corticostriatal neurons.

Homologous desensitization of muscarinic acetylcholine receptors (mAChR) was studied using primary cultures of corticostriatal neurons from neonatal rats. Prolonged incubation with carbachol attenuated phospholipase C responsiveness to muscarinic agonists and decreased the number of cell surface mAChR, as measured by binding of N-[3H] methylscopolamine to neuronal monolayers. When neurons were exposed to carbachol for 15 min, 40% of the mAChR lost from the membrane domain was recovered in the cytosol; a decrease of the total neuronal receptors was detected following an incubation with the agonist lasting longer than 15 min. Both 8-Br-cyclic AMP and forskolin neither affected N-[3H]methylscopolamine binding to cell monolayers or did they prevent the agonist-mediated mAChR desensitization. 8-Br-cyclic GMP also failed to decrease mAChR number. Pertussis toxin failed to prevent the homologous desensitization of mAChR under conditions that blocked the agonist-mediated inhibition of forskolin-stimulated cyclic AMP formation. The phorbol ester 12-O-tetradecanoyl-phorbol-12, 13-acetate induced a concentration-dependent decrease of N-[3H]methylscopolamine binding to neuronal monolayers. However, the protein kinase C inhibitors sphingosine and the ganglioside monosialosyl-gangliotetraglicosylceramide inhibited the 12-O-tetradecanoyl-phorbol-12,13-acetate-induced but not the agonist-induced desensitization of mAChRs. Furthermore, incubation with muscarinic agonists failed to translocate protein kinase C from cytosol to plasma membranes, as measured by binding of the phorbol ester [3H]-4-beta-phorbol-12,13-dibutyrate to neuronal monolayers. In corticostriatal neurons the agonist-induced desensitization and internalization of mAChR involves neither protein kinase C and protein kinase A activation nor changes in cyclic GMP and cyclic AMP content.

Cross-linking of human [125I]beta-endorphin to opioid receptors in rat striatal membranes: biochemical evidence for the existence of a mu/delta opioid receptor complex.

In a modified Krebs buffer at 37 degrees C, the selective mu agonist [3H] D-Ala2,MePhe4,Gly-ol5]enkephalin [( 3H]DAMGO) and the nonselective mu/delta agonist human [125I]beta-endorphin [( 125I]beta-endH) bound to rat striatal membranes with a Kd of about 7 and 5 nM and a Bmax of about 95 and 260 fmol/mg of protein, respectively, consistent with labeling of mu receptors by the former ligand and labeling of both mu and delta receptors by the latter. The binding of 2 nM [125I]beta-endH was displaced by unlabeled DAMGO (IC50 30 nM), [D-Ala2-D-Leu5]enkephalin (IC50 60 nM) as well as by the selective delta agonists [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET, IC50 500 nM) and Tyr-Ala-Phe-Asp-Val-Val-Gly-NH2 (IC50 700 nM) in a monophasic manner within 2 to 3 log concentration units, suggesting an allosteric interaction between mu and delta sites labeled by [125I]beta-endH under these conditions. Accordingly, 500 nM DSTBULET caused almost 40% inhibition of the apparent Bmax without changing the apparent Kd of [3H] DAMGO. The kappa agonist U 50,488 was ineffective as competing ligand even at a concentration of 10 microM. Upon affinity cross-linking of [125I]beta-endH (2 nM) to rat striatal mu- and delta-opioid receptors, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized tissue under reducing conditions followed by autoradiography of the dried gels revealed a major broad band of covalently labeled protein with an apparent molecular weight of 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of 2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid (aminoethylcysteine ketimine) in the bovine brain.

2H-1,4-Thiazine-5,6-dihydro-3-carboxylic acid (trivial name: aminoethylcysteine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts by means of three different procedures. Gas liquid chromatography of protein-free extracts of five bovine brains revealed the presence of this compound at concentrations ranging from 2 to 3 nmol/g wet weight of tissue. The enzymatic method based on the inhibition of D-amino acid oxidase activity by aminoethylcysteine ketimine together with an high-performance liquid chromatography procedure confirm the identification and quantitations obtained with gas liquid chromatography. The discovery of this compound structurally similar to pipecolic acid opens the question of its physiological role in the central nervous system.

Complete purification of two identical Na(+)-pump inhibitors isolated from bovine hypothalamus and hypophysis.

We have completely purified, in parallel, a low molecular weight, non-specific, non-lipidic, Na+,K(+)-ATPase inhibitory factor from bovine hypothalamic and pituitary tissues. In the final purification step we obtain, from both tissues, a single, homogeneous peak, with a maximal absorbance at 247 nm. This factor, at physiological concentrations of potassium (5-25 mM), inhibits in a dose-response manner Na+,K(+)-ATPase and displaces ouabain from its receptor at the enzyme structure. The factor isolated from both tissues is identical, being the specific activity per weight of tissue higher in hypophysis. No factor was found in cerebral cortex, used as tissue control.

[Effects of piracetam on pain sensitivity and levels of beta-endorphin in blood and cAMP in the cerebral cortex of rats]. / Vliianie piratsetama na bolevuiu chuvstvitel'nost', uroven' beta-éndorfina v krovi i tsAMF v kore golovnogo mozga krys.

It has been shown, that piracetam by i.p. injection (10, 100, 400 mg/kg) or by consumption with drinking water 10, 100, 1000 mg/kg/day during 12 days delays extinction of conditioned reflex of passive avoidance and dose-dependence elevates exploratory activity in "open field", pain sensitivity threshold reduces to 70-75%. Prolonged consumption of 1000 mg/kg/day piracetam results in 3-fold decrease of beta-endorphin concentration in plasma (p less than 0.01) and 100 mg/kg/day--in 35% increase of cAMP content in rat brain cortex.