Models of β-amyloid induced Tau-pathology: the long and “folded” road to understand the mechanism

The amyloid cascade hypothesis has been the prevailing hypothesis in Alzheimer’s Disease research, although the final and most wanted proof i.e. fully successful anti-amyloid clinical trials in patients, is still lacking. This may require a better in depth understanding of the cascade. Particularly, the exact toxic forms of Aβ and Tau, the molecular link between them and their respective contributions to the disease process need to be identified in detail. Although the lack of final proof has raised substantial criticism on the hypothesis per se, accumulating experimental evidence in in vitro models, in vivo models and from biomarkers analysis in patients supports the amyloid cascade and particularly Aβ-induced Tau-pathology, which is the focus of this review. We here discuss available models that recapitulate Aβ-induced Tau-pathology and review some potential underlying mechanisms. The availability and diversity of these models that mimic the amyloid cascade partially or more complete, provide tools to study remaining questions, which are crucial for development of therapeutic strategies for Alzheimer’s Disease.     

Oxidative protein folding: From thiol–disulfide exchange reactions to the redox poise of the endoplasmic reticulum

This review examines oxidative protein folding within the mammalian endoplasmic reticulum (ER) from an enzymological perspective. In protein disulfide isomerase-first (PDI-first) pathways of oxidative protein folding, PDI is the immediate oxidant of reduced client proteins and then addresses disulfide mispairings in a second isomerization phase. In PDI-second pathways the initial oxidation is PDI-independent. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Strategies and challenges are discussed for determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDI_red:PDI_ox. The preponderance of evidence suggests that the mammalian ER is more reducing than first envisaged. The average redox state of major PDI-family members is largely to almost totally reduced. These observations are consistent with model studies showing that oxidative protein folding proceeds most efficiently at a reducing redox poise consistent with a stoichiometric insertion of disulfides into client proteins. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, this review concludes with an elaboration of a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC–MS–MS methods. Consortia of oxidoreductases that are in redox equilibrium can then be identified and compared to the glutathione redox poise of the ER to gain a more detailed understanding of the factors that influence oxidative protein folding within the secretory compartment.     

Synergic effects of NO and oxygen free radicals in the injury of ischemia-reperfused myocardium——ESR studies on NO free radicals generated from ischemia-reperfused myocardium

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to β-subunit of hemoglobin (β-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginme. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (<1 mmol/L) could protect the heart from the ischemia-reperfusion injury but at higher con     

Dynamic analysis of competition and complementarity for light and N use to understand the yield and the protein content of a durum wheat–winter pea intercrop

In a previous paper [Bedoussac L, Justes E (2009) Plant Soil, doi: 10.1007/s11104-009-0082-2], we showed that intercropping of durum wheat and winter pea increased the yield and protein concentration of durum wheat when early N availability was less than 120 kg N ha^?1. The aim of the present work was to understand these results by analysing intercrop species dynamics for growth, light and N acquisition. A 2-year field experiment was carried out in southwest France with different fertilizer-N levels in order to compare wheat (Triticum turgidum L.) and pea (Pisum sativum L.) grown as sole crops and as an intercrop in a row substitutive design. The advantages of intercropping in low N conditions were due mainly to: (1) better light use (up to 10%), thanks to species dynamic complementarity for leaf area index and height; (2) growth complementarity over time (higher growth rate of wheat until pea flowering and then of pea until wheat flowering); and (3) dynamic complementary N acquisition associated with better wheat N status throughout growth. Disadvantages, underlining poorer complementarity within the intercrop stand, were observed with ample available N in early growth. This induced higher cereal growth during winter, which led to increase interspecies competition by reducing pea light absorption and, consequently, its biomass production.     

Oxidation of α-tocopherol in micelles and liposomes by the hydroxyl,perhydroxyl, and superoxide free radicals

Rates of oxidation of α-tocopherol by the hydroxyl- and superoxide free radicals were measured. The radicals were produced in known yields by radiolysis of aqueous solutions with gamma rays. Two main systems were used to dissolve the tocopherol; micelles, made up from charged and uncharged amphiphiles, and membranes made from dimyristyl phosphatidylcholine which could be charged by addition of stearyl amine or dicetyl phosphate. The HO. radicals were efficient oxidants of α-tocopherol in all systems, with up to 83% of radicals generated in micelle and 32% in membrane suspensions initiating the oxidation. The HO_{$\text{2}\text{?}$} radical was an even more effective oxidant, but when most of it was in the O form at neutral or alkaline pH, the oxidation rates became low. Tocopherol held in positively charged micelles or membranes was oxidized at a higher rate by the O than in uncharged or negative particles. Possible biological significance of these results is discussed.     

Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and “suicide inactivation” of metmyoglobin

BackgroundMetmyoglobin (MbFe^III) reaction with H₂O₂ has been a subject of study over many years. H₂O₂ alone promotes heme destruction frequently denoted “suicide inactivation,” yet the mechanism underlying H₂O₂ dismutation associated with MbFe^III inactivation remains obscure.MethodsMbFe^III reaction with excess H₂O₂ in the absence and presence of the nitroxide was studied at pH 5.3–8.1 and 25 °C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H₂O₂ depletion, O₂ evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy.ResultsThe rates of both H₂O₂ dismutation and heme inactivation processes depend on [MbFe^III], [H₂O₂] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe^III molecule catalyzes the dismutation of 50 ± 10 H₂O₂ molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe^III while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe^IVO by H₂O₂ and by nitroxide, respectively.ConclusionsThe nitroxide effects on H₂O₂ dismutation catalyzed by MbFe^III demonstrate that MbFe^IVO reduction by H₂O₂ is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I–H₂O₂ adduct, which decomposes to a MbFe^IVO and an inactivated heme at a ratio of 25:1.General significanceThe effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.     

Recognition mechanism of Wilms’ tumour suppressor protein and DNA triplets: insights from molecular dynamics simulation and free energy analysis

The Wilms’ tumour suppressor protein (WT1) plays a multifaceted role in human cancer processes. Mutations on its DNA recognition domain could lead to Denys–Drash syndrome, and alternate splicing results in insertion of the tripeptide Lys–Thr–Ser (KTS) between the third and fourth zinc fingers (ZFs), leading to changes in the DNA-binding function. However, detailed recognition mechanisms of the WT1–DNA complex have not been explored. To clarify the mutational effects upon WT1 towards DNA binding at the atomic level, molecular dynamics simulations and the molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) method were employed. The simulation results indicate that mutations in ZF domains (E427Q and Q369H) may weaken the binding affinity, and the statistical analyses of the hydrogen bonds and hydrophobic interactions show that eight residues (Lys351, Arg366, Arg375, Arg376, Lys399, Arg403, Arg424 and Arg430) have a significant influence on recognition and binding to DNA. Insertion of the tripeptide KTS could form an immobilized hydrogen-bonding network with Arg403, affecting the flexibility and angle of the linker between ZF3 and ZF4, thus influencing the recognition between the protein and the DNA triplet at its 5′ terminus. These results represent the first step towards a thorough characterization of the WT1 recognition mechanisms, providing a better understanding of the structure–function relationship of WT1 and its mutants.     

Mapping the protein–protein and genetic interactions of cancer to guide precision medicine

Massive efforts to sequence cancer genomes have compiled an impressive catalogue of cancer mutations, revealing the recurrent exploitation of a handful of ‘hallmark cancer pathways’. However, unraveling how sets of mutated proteins in these and other pathways hijack pro-proliferative signaling networks and dictate therapeutic responsiveness remains challenging. Here, we show that cancer driver protein–protein interactions are enriched for additional cancer drivers, highlighting the power of physical interaction maps to explain known, as well as uncover new, disease-promoting pathway interrelationships. We hypothesize that by systematically mapping the protein–protein and genetic interactions in cancer—thereby creating Cancer Cell Maps—we will create resources against which to contextualize a patient’s mutations into perturbed pathways/complexes and thereby specify a matching targeted therapeutic cocktail.     

Reaction of thiyl radicals with alcohols,ethers and polyunsaturated fatty acids: A possible role of thiyl free radicals in thiol mutagenesis?

Summary Possible reactions of thiyl free radicals in biological environment are reviewed. In particular hydrogen transfer processes from model C–H compounds like alcohols and ethers as well as from polyunsaturated fatty acids to thiyl radicals are described to proceed with reasonably high rate constants (10₃ – 10⁴ and 10⁶ – 10⁷ M^–1 s^–1, respectively). Thiyl radicals have thus to be considered as potentially hazardous species especially with respect to DNA damage and lipid peroxidation.Paper given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.1990     

Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein–DNA binding site

Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein–DNA interactions when compared to a simple linear duplex fragment. The number of protein–DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA.     

Safety of the production process of SURFACEN® to inactivate and remove virus

SURFACEN^® is a biological product produced from pig lungs. Since these animals can be potential sources of microbial pathogens such as viruses, the manufacturing process of this product should guarantee safety from health hazards. The SURFACEN^® production procedure is capable of effective viral clearance (inactivation/removal) by involving two stages of organic solvent extraction followed by acetone precipitation and heat treatment. In this study, we evaluated the clearance capacity of these four stages for a wide range of viruses by performing spiking experiments. Residual contamination was assessed using a Tissue Culture Infectious Dose assay (log₁₀ TCID₅₀). The validation study demonstrated that, for all viruses tested, the TCID₅₀ titers were reduced by more than 2 log₁₀ in each stage. Total log reduction values achieved were between ≥17.82 log₁₀ and ≥27.93 log₁₀, depending on the virus physical properties, titer, and the number of processing stages applied. Results indicated that the production procedure of SURFACEN^® can inactivate or remove contaminant viruses from the raw material.     

DNA damage in human colonic mucosa cells evoked by nickel and protective action of quercetin - involvement of free radicals?

Nickel is a toxic and carcinogenic environmental and occupational pollutant and quercetin is a dietary flavonoid that is reported to modulate effects of many mutagens and carcinogens. We investigated the ability of nickel chloride to induce DNA damage in human colonic mucosa cells in the presence of quercetin, using the alkaline comet assay. Nickel chloride (5–250 μmol/L) evoked dose-dependent DNA damage and quercetin at 50 μmol/L decreased the extent of this damage. The cells exposed to nickel chloride progressively removed their DNA damage and the presence of 50 μmol/L quercetin in the repair-incubation medium did not affect the repair kinetics. Cells exposed to nickel and treated with endonuclease III, an enzyme recognizing oxidized bases, displayed a greater extent of DNA damage than those not treated with the enzyme. Quercetin did not exert a significant effect on the production of oxidized bases by nickel. Pretreatment of the cells with a nitrone spin trap, N-tert-butyl-α-phenylnitrone, decreased the extent of DNA damage evoked by nickel. Quercetin caused a further decrease in the extent of the damage in the presence of the trap. The results obtained suggest that reactive oxygen species, including free radicals, might be involved in the formation of DNA lesions induced by nickel chloride in colonic mucosa cells and that quercetin may exert protective effects in these cells. This revised version was published online in July 2006 with corrections to the Cover Date.