α2-Macroglobulin as a β-Amyloid Peptide-Binding Plasma Protein

Abstract: The β-amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α₂-macroglobulin (α₂M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds ¹²⁵I-Aβ_(1–42) with high affinity (apparent dissociation constant of 3.8 × 10^?10M). Approximately 1 mol of Aβ is bound per mole of α₂M. Both native and methylamine-activated α₂M bind ¹²⁵I-Aβ_(1–42). The binding of ¹²⁵I-Aβ_(1–42) to α₂M is enhanced by micromolar concentrations of Zn²⁺ (but not Ca²⁺) and is inhibited by noniodinated Aβ_(1–42) and Aβ_(1–40) but not by the reverse peptide Aβ_(40-1) or the cytokines interleukin 1β or interleukin 2. α₁-Antichymotrypsin, another plaque-associated protein, inhibits both the binding of ¹²⁵I-Aβ_(1–42) to α₂M as well as the degradation of ¹²⁵I-Aβ_(1–42) by proteinase-activated α₂M. Moreover, the binding of ¹²⁵I-Aβ_(1–42) to α₂M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α₂M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.     

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.     

Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate- zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.      

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Temperature and benzyl alcohol as probes of the antilipolytic mechanism of insulin action in adipocytes.

The temperature dependence of cAMP accumulation and glycerol release in response to epinephrine and insulin in adipocytes is examined. (1) Glycerol release in the presence of epinephrine demonstrated linear Arrhenius kinetics to 41 degrees C, and above 45 degrees C glycerol release was progressively inhibited. (2) In contrast, incubation of the cells with both epinephrine and insulin resulted in glycerol release rates that were relatively temperature insensitive. (3) Calculation of the efficacy of insulin to inhibit epinephrine-stimulated glycerol release as a function of temperature yielded a biphasic response, with a distinct optimum around 41 degrees C, in a similar manner to the effects of insulin on hexose transport activation determined previously. (4) A saturating dose of insulin (40 ng/ml) was found to have no significant effect on epinephrine-stimulated intracellular cAMP over the temperature range studied. (5) Addition of benzyl alcohol (to 40 mM) resulted in substantial inhibition of basal, epinephrine stimulated, and insulin inhibited glycerol release, without affecting the magnitude of insulin inhibition. We conclude from these studies that (a) insulin inhibition of glycerol release can not be mediated directly by intracellular cAMP modulation, (b) as in the case of hexose transport activation, the signalling mechanism by the occupied insulin receptor appears to be relatively independent of the membrane lipid environment.     

Organization and interaction of cholesterol and phosphatidylcholine in model bilayer membranes

The molecular organization of sterols in liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 37 degrees C is examined by utilizing the fluorescent analogue of cholesterol cholesta-5,7,9-trien-3 beta-ol (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:1 mol/mol) at various cholesterol/cholestratrienol molar ratios (0.5:1 up to 1:1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration dependence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1:1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:1. (6) From qualitative calculations of the van der Walls' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes.     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

Neuromuscular and phospholipase activities of venoms from three subspecies of Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus)

The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. In this work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus) of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipase A2 (PLA2) activities and electrophoretic profiles. The order of potency of PLA2 activity was B. n. diporus>B. n. paranaensis>B. n. goyazensis. In BC preparations, B. n. goyazensis venom (50 microg/mL) was significantly (p<0.05) more active than B. n. paranaensis and B. n. diporus venoms, which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms (100 microg/mL) produced blockades of 57.4+/-5%, 30+/-3% and 17.4+/-7% (n=3-6 each), respectively. The three venoms inhibited contractures in response to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a long-lasting, concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responses within 45-100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA2 activity and the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among the venoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship with PLA2 activity varied with the preparation used.