Structural basis for the recognition and cross-linking of amyloid fibrils by human apolipoprotein E

Apolipoprotein (apo) E is a well characterized lipid-binding protein in plasma that also exists as a common nonfibrillar component of both cerebral and systemic amyloid deposits. A genetic link between a common isoform of apoE, apoE4, and the incidence of late onset Alzheimer disease has drawn considerable attention to the potential roles of apoE in amyloid-related disease. We examined the interactions of apoE with amyloid fibrils composed of apoC-II and the amyloid-beta (Abeta) peptide. Aggregates of apoE with Abeta and apoC-II are found in Alzheimer and atherosclerotic plaques, respectively. Sedimentation velocity and fibril size distribution analysis showed that apoE3 and E4 isoforms bind and noncovalently cross-link apoC-II fibrils in a similar manner. This ability to cross-link apoC-II fibrils was abolished by the dissociation of the apoE tetramer to monomers or by thrombin cleavage to yield separate N- and C-terminal domains. Preparative ultracentrifuge binding studies indicated that apoE and the isolated N- and C-terminal domains of apoE bind with submicromolar affinities to both apoC-II and Abeta fibrils. Fluorescence quenching and resonance energy transfer experiments confirmed that both domains of apoE interact with apoC-II fibrils and demonstrated that the binding of the isolated N-terminal domain of apoE to apoC-II or Abeta fibrils is accompanied by a significant conformational change with helix three of the domain moving relative to helix one. We propose a model involving the interaction of apoE with patterns of aligned residues that could explain the general ability of apoE to bind to a diverse range of amyloid fibrils.     

A simple plant regeneration-ability assay in a range of Lycopersicon species

Plant growth regulator-dependent (PGR-dependent) in vitro shoot organogenesis has been extensively studied in tomato (Lycopersicon esculentum), whereas PGR-independent adventitious shoot organogenesis received marginal attention in L. esculentum and no consideration at all in other Lycopersicon species. In the present study, induction of PGR-independent adventitious shoots was by decapitation of the apex and removal of preexisting shoot meristems of the seedling, and seedling culture on a medium with no PGR supplements. The existence of PGR-independent regeneration-ability was verified in L. esculentum genotypes (high pigment photomorphogenic mutants and wild-type counterparts) and was uncover amongst L. cheesmanii, L. chilense, L. chmielewskii, L. hirsutum, L. parviflorum, L.␣peruvianum and L. pimpinellifolium. Compared to species other than L. esculentum, high pigment photomorphogenic mutants displayed the weakest PGR-independent regeneration-ability. Our results imply that decapitated seedlings cultured on a medium without PGRs can serve as a convenient assay system for genotypic variation in self-controlled, PGR-independent, shoot regeneration-ability in a wide range of Lycopersicon species. Using transverse thin slices of the hypocotyl placed onto a medium supplemented with 0.2 μM zeatin reboside and 0.04 μM IAA, we assessed PGR-mediated shoot regeneration in L. esculentum genotypes. In a given genotype, more plants per seedling were established by PGR-mediated than by PGR-independent regeneration. However, with both modes of organogenesis, only a fraction of shoot buds eventually grew into normal plants, while others developed into abnormal regenerants having no stem. Percentage of stem-deficiency, in a given genotype, was higher in PGR-treated cultures, which indicates that PGRs amplify the formation frequency of imperfect adventitious apical shoot meristems. Unlike L. esculentum, adventitious shoot buds of other Lycopersicon species, induced by wounding seedlings that were not treated with PGRs, rarely formed regenerants lacking a stem.     

Arrestin binding to calmodulin: a direct interaction between two ubiquitous signaling proteins

Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.     

A Neurotrophic Factor Derived from Goldfish Brain: Characterization and Purification

Previous studies carried out in our laboratory have demonstrated that goldfish brain contains substances that promote neurite extension from regenerating retinae in culture. Fractionation of the brain extract by molecular sieving chromatography revealed the presence of several molecular species, including two peaks that have neurotrophic activity, representing low-molecular-weight substances. One peak was eluted (P-a) with an apparent molecular weight of about 13 kDa and was designated substratum neurite extension factor (SNEF) because it retained its neurotrophic activity when adsorbed onto the substratum. This recovered Sephadex fraction (P-a) when applied in vivo intraocularly caused an earlier capacity of the corresponding retinae to sprout in vitro. Thus, at 3 and 5 days after injury the neuritic growth indices from the factor-treated retinae were of 0.9 +/- 0.2 and 2.8 +/- 0.5, respectively, as compared with indices of 0.3 +/- 0.1 and 0.9 +/- 0.2, respectively, in retinae of injured but nontreated nerves. The factor was further purified by two steps of HPLC (ion exchange followed by reversed phase). The results showed that it is an acidic glycoprotein with an apparent molecular weight of 10 kDa.     

Diffusional boundary spreading in the ultracentrifuge

A common approximation for deriving solutions to the Lamm equation is to neglect diffusion. This paper presents a singular perturbation technique that allows one to estimate the band spreading due to nonzero diffusion coefficient. We illustrate the general mathematical technique by its application to sedimentation when pressure effects are important. Comparison of the approximate solution with accurate numerical solutions shows that the relative errors are of the order of 1% both for concentration and concentration gradient for parameters of chemical interest.     

The steady state activity of succinate dehydrogenase in the presence of opposing effectors

The extent of the deactivation of the mitochondrial succinate dehydrogenase by oxaloacetate is a function of the redox state of the enzyme. Oxidized enzyme is deactivated by much lower concentrations of oxaloacetate than those needed to deactivate reduced enzyme. An accurate method for measuring this relationship is the redox titration of the enzymic activity of succinate dehydrogenase, carried out in the presence of oxaloacetate. For each concentration of oxaloacetate a different redox titration curve was reported with the apparent mid-potential decreasing with increasing oxaloacetate. These results are compatible with a model which proposes that both oxidized and reduced enzymes can form the catalytically non-active complex with oxaloacetate, but that the complex formed the the oxidized enzyme is more stable than that formed by the reduced enzyme. When the oxaloacetate concentration is low, reduction of the enzyme will lower the fraction of the succinate dehydrogenase-oxaloacetate complex, a reaction which we observe as reductive activation of the enzyme. If this experiment is repeated in the presence of high concentration of oxaloacetate, no activation of the enzyme takes place, but the low stability of the reduced enzyme oxaloacetate complex is revealed by the rapid exchange of the enzyme-bound oxaloacetate with the free ligand. The rate of this exchange is extremely slow at high positive potential and becomes faster upon lowering of the poise potential. The reductive activation of the succinate dehydrogenase is regarded as a two step reaction. In the first step the reduced non-active complex releases the oxaloacetate and in the second step the active form of the enzyme is evolved. These two steps can be observed experimentally; Reductive activation at a redox potential higher than the mid-potential of the oxaloacetate-malate couple (minus 166 mV) is characterized by Ea = 18 Kca/mole, the final equilibrium level of activation decreases upon lowering of the temperature. Reduction activation of the enzyme at minus 240 mV is a very rapid reaction which goes to completion at all temperatures tested and has an activation energy of 12.5 Kcal/mole. The mechanism of the reductive activation and its possible role in the regulation of succinate dehydrogenase in the mitochondria is discussed.     

Modulation of mitochondrial succinate dehydrogenase activity,mechanism and function

Summary The mitochondrial succinate dehydrogenase (E.C. 1.3.3.99) is subjected to apparently complicated regulatory mechanism. Yet, systematic analysis of the mechanism reveals the simplicity of the control. There are two stable forms of the enzyme; the non-active form stabilized as 1:1 complex with oxaloacetate and the active form stabilized by binding of activating ligands. This model quantitatively describes either the equilibrium level of active enzyme or the kinetics of activation-deactivation, in the presence of various concentrations of opposing effectors. The site where the regulatory ligands interact with the enzyme is not the substrate bonding site. The marked differences of dissociation constants of the same ligand from the two sites clearly distinguish between them.This model is fully developed for simple cases where the activating ligands are dicarboxylic acids or monovalent anions. On the other hand with activators such as ATP or CoQH₂, quantitation is still not at hand. This stems from the difficulties in maintaining determined, measurable, concentrations of the ligand in equilibrium with the membranal enzyme.While in active form the histidyl flavin moity of the enzyme is reduced by physiological substrate (succinate; CoQH₂). The non-active form is not reduced by these compounds, only strong reductants with low redox potential reduce the non-active enzyme. It is suggested that deactivation is a simple modulation of the redox potential of the flavin form E 0 mV in the active enzyme to E < –190 mV. The switch from one state to another might be achieved by distortion of the planar form of oxidized flavin to the bend configuration of the reduced flavin. Thus, in the active enzyme such distortion will destabilize the oxidized state of the flavin, shifting the redox potential to the higher value. The binding of oxaloacetate to the regulatory sites releases the distorting forces by relaxing the conformation of the enzyme. Consequently, the flavin assumes its planar form with the low redox potential. This assumption is supported by the spectral shifts of the flavin associated with the activation deactivation transition.The suicidal oxidation of malate to oxaloacetate, carried by the succinate dehydrogenase, plays an important role in modulating the enzyme activity in the mitochondria. This mechanism might supply oxaloacetate for deactivation in spite of the negligible concentration of free oxaloacetate in the matrix. The oxidation of malate by the enzyme is controlled by the redox potential at the immediate vicinity of the enzyme, and is imposed by the redox level of the membranal quinone.Finally, the modulation of succinate dehydrogenase activity is closely associated with regulation of NADH oxidation through the mutual inhibition between oxidases (Gutman, M. in Bioenergetics of Membranes, L. Packer et al., ed. Elsevier 1977, p. 165). The consequence of these interactions is the selection for the main electron donnor for the respiratory chain, during mixed substrate respiration, according to the metabolic demands from the mitochondria.Abbreviations SDH succinate dehydrogenase (succinate: acceptor oxidoreductase (E.C. 1.3.99.1)); - OAA oxaloacetate - Act activator - E_A, E_A active and non active forms of the enzyme, respectively - K'eq apparent equilibrium constant - K'd apparent dissociation constant - K_Act, K_OAA dissociation constant of the respective ligand from the enzyme - K'a, k'd the apparent rate constants of activation and deactivation, respectively - ka, kd the true rate constant of activation and deactivation respectively - ETP, ETP_II non phosphorylating and phosphorylating submitochondrial particles - PMS phenazine methosulfate - DCIP dichlorophenol indophenol - CoQ ubiquinone - TIFA Thenotriflouvoacetone - NEM N methyl Maleimide     

Prenatal diagnosis of familial hypercholesterolemia caused by the “Lebanese” mutation at the low density lipoprotein receptor locus

Summary Here, we report the prenatal diagnosis of familial hypercholesterolemia in a Christian-Arab family that carries the Lebanese mutation, a single base substitution that creates a HinfI restriction site, at the low density lipoprotein (LDL) receptor locus. Polymerase chain reaction amplification and restriction analysis were performed on genomic DNA extracted from a chorionic villus sample. In conjunction with karyotype analysis, the fetus was identified as a heterozygous female. Analysis of LDL receptor restriction fragment length polymorphisms confirmed the presence of a male parent marker and revealed that the fetus inherited the mutant gene from its mother. This technique offers a simple and rapid diagnostic tool that can be carried out at an early stage of gestation. It is recommended for families and population groups with molecularly defined LDL receptor mutations.