APP Expression in Primary Neuronal Cell Cultures fromP6 Mice during in vitro Differentiation

Primary neuronal cell cultures from P6 mice were investigated in order to study amyloid protein precursor (APP) gene expression in differentiating neurons. Cerebellar granule cells which strongly express APP 695 allowed the identification of three distinct isoforms of neuronal APP 695. The high-molecular-weight form of APP 695 is sialylated. The expression pattern of neuronal APP 695 changes during in vitro differentiation. Sialylated forms become more abundant upon longer cultivation time. The secreted forms of sialylated, neuronal APP 695 are shown to comigrate with APP isolated from cerebrospinal fluid. We suggest that the different sialylation states of APP 695 may reflect the modulation of cell-cell and cell-substrate interactions during in vitro differentiation and regeneration

Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis

Glyoxalase II (GloII) is a ubiquitous thioester hydrolase catalyzing the last step of the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Here, we present a detailed structure-function analysis of cGloII from the malaria parasite Plasmodium falciparum. The activity of the enzyme was salt-sensitive and pH-log k(cat) and pH-log k(cat)/K-m profiles revealed acid-base catalysis. An acidic pK(a)(app) value of approximately 6 probably reflects hydroxide formation at the metal center. The glutathione-binding site was analyzed by site-directed mutagenesis. Substitution of residue Arg(154) caused a 2.5-fold increase of K-m(app), whereas replacements of Arg(257) or Lys(260) were far more detrimental. Although the glutathione-binding site and the catalytic center are separated, six of six single mutations at the substrate-binding site decreased the k(cat)(app) value. Furthermore, product inhibition studies support a Theorell-Chance Bi Bi mechanism with glutathione as the second product. We conclude that the substrate is predominantly bound via ionic interactions with the conserved residues Arg(257) and Lys(260), and that correct substrate binding is a pH-and salt-dependent rate-limiting step for catalysis. The presented mechanistic model is presumably also valid for GloII from many other organisms. Our study could be valuable for drug development strategies and enhances the understanding of the chemistry of binuclear metallohydrolases

Microstructured superhydrorepellent surfaces: Effect of drop pressure on fakir-state stability and apparent contact angles

In this paper we present a generalized Cassi-Baxter equation to take into account the effect of drop pressure on the apparent contact angle theta_{app}. Also we determine the limiting pressure p_{W} which causes the impalement transition to the Wenzel state and the pull-off pressure p_{out} at which the drop detaches from the substrate. The calculations have been carried out for axial-symmetric pillars of three different shapes: conical, hemispherical topped and flat topped cylindrical pillars. Calculations show that, assuming the same pillar spacing, conical pillars may be more incline to undergo an impalement transition to the Wenzel state, but, on the other hand, they are characterized by a vanishing pull-off pressure which causes the drop not to adhere to the substrate and therefore to detach very easily. We infer that this property should strongly reduce the contact angle hysteresis as experimentally osberved in Ref. \cite{Martines-Conical-Shape}. It is possible to combine large resistance to impalement transition (i.e. large value of p_{W}) and small (or even vanishing) detaching pressure p_{out} by employing cylindrical pillars with conical tips. We also show that depending on the particular pillar geometry, the effect of drop pressure on the apparent contact angle theta_{app} may be more or less significant. In particular we show that in case of conical pillars increasing the drop pressure causes a significant decrease of theta_{app} in agreement with some experimental investigations \cite{LafunaTransitio}, whereas theta_{app} slightly increases for hemispherical or flat topped cylindrical pillars.Comment: 21 pages, 13 figure

Chemical order and crystallographic texture of FePd:Cu thin alloy films

FePd thin films have been recently considered as promising material for high-density magnetic storage devices. However, it is necessary to find a proper method of fabrication for the (001)-textured and chemically well-ordered alloy. In this paper, we present the detailed investigations of lattice parameters, chemical order degree, grain sizes and crystallographic texture, carried out on FePd alloys with 10 at.% of Cu addition. The initial [Cu(0.2 nm)/Fe(0.9 nm)/Pd(1.1 nm)]x5 multilayers were thermally evaporated in an ultra-high vacuum on MgO(100), Si(100), Si(111) and Si(100) covered by 100 nm thick layer of amorphous SiO2. In order to obtain homogeneous FePd:Cu alloy, the multilayers were annealed in two different ways. First, the samples were rapidly annealed in nitrogen atmosphere at 600oC for 90 seconds. Next, the long annealing in a high vacuum for 1 hour at 700oC was done. This paper focuses on quantitative investigations of the chemical order degree and crystallographic texture of ternary FePd:Cu alloys deposited on four different substrates. In order to obtain both quantities we have taken a novel approach to consider the problem of dopant atoms located in the FePd structure. The studies of the structure were done using X-Ray Diffraction (XRD) performed with synchrotron radiation and pole figures measurements. We have found that the addition of Cu changes the FePd lattice parameters and lattice distortion. We have also shown, that using different substrates it is possible to obtain a FePd:Cu alloy with different chemical order and texture. Moreover, it was observed that texture category is substrate dependent

Control of the colossal magnetoresistance by strain effect in Nd0.5_{0.5}Ca0.5_{0.5}MnO3_{3} thin films

Thin films of Nd$_{0.5}$Ca$_{0.5}$MnO$_{3}$ manganites with colossal magnetoresistance (CMR) properties have been synthesized by the Pulsed Laser Deposition technique on (100)-SrTiO$_{3}$. The lattice parameters of these manganites and correlatively their CMR properties can be controlled by the substrate temperature $T_{S}$. The maximum CMR effect at 75K, calculated as the ratio $\rho (H=0T)/\rho (H=7T)$ is 10$^4$ for a deposition temperature of $T_{S}=680$ degC. Structural studies show that the Nd$_{0.5}$Ca$_{0.5}$MnO$_{3}$ film is single phase, [010]-oriented and has a pseudocubic symmetry of the perovskite subcell with a=3.77$\AA$ at room temperature. We suggest that correlation between lattice parameters, CMR and substrate temperature $T_{S}$ result mainly from substrate-induced strains which can weaken the charge-ordered state at low temperature.Comment: 9 pages, 4 figures. To be published in Applied Physics Letter

Influence of the electron-phonon interfacial conductance on the thermal transport at metal/dielectric interfaces

Thermal boundary conductance at a metal-dieletric interface is a quantity of prime importance for heat management at the nanoscale. While the boundary conductance is usually ascribed to the coupling between metal phonons and dielectric phonons, in this work we examine the influence of a direct coupling between the metal electrons and the dielectric phonons. The effect of electron- phonon processes is generally believed to be resistive, and tends to decrease the overall thermal boundary conductance as compared to the phonon-phonon conductance {\sigma}p . Here, we find that the effect of a direct coupling {\sigma}e is to enhance the effective thermal conductance, between the metal and the dielectric. Resistive effects turn out to be important only for thin films of metals having a low electron-phonon coupling strength. Two approaches are explored to reach these conclusions. First, we present an analytical solution of the two-temperature model to compute the effective conductance which account for all the relevant energy channels, as a function of {\sigma}e , {\sigma}p and the electron-phonon coupling factor G. Second, we use numerical resolution to examine the influence of {\sigma}e on two realistic cases: gold film on silicon or silica substrates. We point out the implications for the interpretation of time-resolved thermoreflectance experiments

Different types of γ-secretes complexes and their effect on substrate processing

The γ-secretase complex is a transmembrane aspartyl protease that generates the Alzheimer disease (AD) related amyloid β-peptide (Aβ) from the amyloid precursor protein (APP). The γ- secretase complex cleaves APP at two different sites (γ- and ε-sites) generating Aβ-peptides and the APP intracellular domain (AICD). The Aβ-peptide can vary in length, where the most common lengths are of 40 or 42 residues (Aβ40 and Aβ42). The longer Aβ42 peptide is more hydrophobic and prone to aggregate into toxic oligomers. These oligomers will eventually form the extracellular plaques, which are one of the hallmarks found in the brain of AD patients. The γ-secretase complex processes many other substrates besides APP. One important substrate is the Notch receptor that is crucial for critical signaling and cell fate decisions. The failure of γ-secretase inhibitors used in clinical trials can partly be explained by the large number of substrates. Most of these inhibitors give severe side effects related to the impairment of the Notch signaling pathway. Therefore, it is essential to identify strategies to affect the APP processing without disturbing the processing of other substrates. The γ- secretase complex is composed of four components; Presenilin (PS), Nicastrin (Nct), Anterior pharynx defective-1 (Aph-1), and Presenilin enhancer-2 (Pen-2). Both PS and Aph-1 exist as two homologues (PS1/PS2 and Aph-1a/Aph1b) and the Aph-1a homologue can also undergo alternative splicing generating a short (Aph-1aS) or a long (Aph-1aL) isoform. Thus, the different homologues and splice variants can generate up to six distinct γ-secretase complexes with possible diverse functions. In addition, the γ-secretase complex can also undergo caspase cleavage, which may change the properties of the complex. Inhibiting or modulating certain types of γ-secretase complexes could be one way to avoid severe side effects. The general aim of this thesis is therefore to achieve a more detailed understanding of the different γ- secretase complexes and their components, with respect to their properties and substrate selectivity. In Paper I, we reported that single residues in a γ-secretase component besides presenilin, such as Nicastrin, affected the processing of γ-secretase substrates differently. In Paper II, we examined how γ-secretase processing of APP and Notch was affected by the caspase cleavage of PS1. We found that caspase-cleaved γ-secretase complexes still could process APP and Notch, but with an increased intracellular Aβ42/Aβ40 ratio. In Paper III, we investigated whether PS1 and PS2 show different substrate specificity by analyzing the processing of the γ- secretase substrates APP, Notch, N-cadherin, and ephrinB. We found that while the PS1 depletion affected the cleavage of all substrates, the effect of PS2 deficiency was minor. In the final study, Paper IV, we found that whereas γ-secretase complexes containing either Aph-1a or Aph-1b processed APP and Notch to the same extent, they showed different preference of forming complexes with the PS proteins. Aph-1a favored PS1-containing complexes, while Aph-1b rather was incorporated into PS2-containing complexes. All together, these findings support the existence of different active γ-secretase complexes and their possible diverse effects on substrate processing

The Nicastrin ectodomain adopts a highly thermostable structure

Nicastrin is a type I transmembrane glycoprotein, which is part of the high molecular weight gamma-secretase complex. gamma-Secretase is one of the key players associated with the generation of Alzheimer's disease pathology, since it liberates the neurotoxic amyloid beta-peptide. Four proteins Nicastrin, anterior pharynx-defective-1 (Aph-1), presenilin enhancer-2 (Pen-2) and Presenilin are essential to form the active gamma-secretase complex. Recently it has been shown, that Nicastrin has a key function in stabilizing the mature gamma-secretase complex and may also be involved in substrate recognition. So far no structural data for the Nicastrin ectodomain or any other gamma-secretase component are available. We therefore used Circular Dichroism (CD) spectroscopy to demonstrate that Nicastrin, similar to its homologues, the Streptomyces griseus aminopeptidase (SGAP) and the transferrin receptor (TfR), adopts a thermostable secondary structure. Furthermore, the Nicastrin ectodomain has an exceptionally high propensity to refold after thermal denaturation. These findings provide evidence to further support the hypothesis that Nicastrin may share evolutionary conserved properties with the aminopeptidase and the transferrin receptor family

The Alzheimer’s disease γ-secretase generates higher 42:40 ratios for β-amyloid than for p3 peptides

Alzheimer’s disease is characterized by intracerebral deposition of β-amyloid (Aβ). While Aβ40 is the most abundant form, neurotoxicity is mainly mediated by Aβ42. Sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases gives rise to full-length Aβ (Aβ1-x) and N-terminally truncated Aβ′ (Aβ11-x) whereas cleavage by α- and γ-secretases leads to the shorter p3 peptides (Aβ17-x). We uncovered significantly higher ratios of 42- versus 40-ending variants for Aβ and Aβ′ than for p3 secreted by mouse neurons and human induced pluripotent stem cell (iPSC)-derived neurons or produced in a cell-free γ-secretase assay with recombinant APP-CTFs. The 42:40 ratio was highest for Aβ′, followed by Aβ and then p3. Mass spectrometry analysis of APP intracellular domains revealed differential processing of APP-C83, APP-C89, and APP-C99 by γ-secretase already at the ε-cleavage stage. This mechanistic insight could aid in developing substrate-targeted modulators of APP-C99 processing to specifically lower the Aβ42:Aβ40 ratio without compromising γ-secretase function