Preservation of native conformation during aluminium-induced aggregation of tau protein

ALUMINIUM exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27(Al) NMR spectroscopy. The CD studies revealed a five-fold increase in the observed ellipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. Al-27 NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits,the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for tills process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.

A simple model for protein thermal denaturation

Whether proteins denature in all-or-none fashion or in a continuous fashion is as yet an unresolved problem. The all-or-none process implies that while the process of denaturation is going on, only two kinds of protein molecules can exist. One is completely unchanged and the other is altered. The altered protein molecules are indistinguishable. Underlying the 'continuum' models is the assumption that all the chains in a protein globule undergo similar changes so that it is enough to consider a single chain.

Structure and Function of Hemoglobin Confined Inside Silica Nanotubes

Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 degrees C as compared with that of free Hb in solution.

Reproduction and population structure in European aspen

The European aspen (Populus tremula) is a keystone species for biodiversity in boreal forests. However, the future of aspen may be threatened, because large aspens have mostly been removed from managed forests, whereas regeneration and the long-term persistence of mature trees are subjects of concern in protected areas. Aspen is a pioneer tree, and it can reproduce both sexually by seed and asexually by root suckers. Through asexual reproduction aspen forms clones, groups of genetically identical trees (ramets). In my thesis, I have studied the structure of aspen populations in terms of number, size, clonal and demographic properties. Additionally, I have investigated the emergence and survival of seedlings as well as the seed quantity and quality in crosses between the European and hybrid aspen. To study the regeneration and population structure, mature aspens were recorded in old-growth and managed forests in eastern Finland based on a large-scale inventory (11 400 ha). In addition, small aspen trees were surveyed on sample plots. Clonal structure was investigated both by morphological characters and by DNA-based markers (microsatellites). Seedling emergence and survival was studied with two sowing experiments. With crosses between European and hybrid aspens we wanted to study whether elevated temperatures due to climate change would benefit the different crosses of European and hybrid aspen unequally and thus affect the gene flow between the two species. The average volumes of mature aspen were 5.3 m3/ha in continuous old-growth, and 0.8 m3/ha in managed forests. Results indicate also that large aspen trees in managed forests are a legacy of the past less intensively managed forest landscapes. Long-term persistence of aspen in protected areas can only be secured by restoration measures creating sufficiently large gaps for regeneration. More emphasis should be given to sparing aspens in thinnings and to retaining of mature aspens in regeneration cutting in managed forests. Aspen was found to be spatially aggregated in the landscape. This could be explained by site type, disturbance history and / or limitations in seed dispersal. Clonal structure does not explain the spatial aggregation, since average size of the clones was only 2.3 ramets, and most clones (70 %) consisted of just one ramet. The small size of the clones suggests that most of them are relatively young. Therefore, sexual reproduction may be more common than has previously been thought. Seedling emergence was most successful in mineral soil especially, when the site had been burned. Only few seedlings occurred on humus. Survival of the seedlings was low, and strongly dependent on moisture, but also on seedbed conditions. The seeds were found to maintain their germinability longer than has earlier been thought to be possible. Interspecific crosses produced more seeds with higher quality than intraspecific crosses. When temperature was elevated, germination of hybrid aspen seeds increased more than seeds from P. tremula x P. tremula crosses. These results suggest that hybrid aspen may have a significant genetic impact on the European aspen, and this effect may become strengthened by climate warming.

Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac and parecoxib

Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.

Essential Thrombocythaemia : Diagnosis, Prognostic Aspects, and the Outcome of Finnish patients

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) characterized by thrombocytosis, i.e. a constant elevation of platelet count. Thrombocytosis may appear in MPDs (ET, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis) and as a reactive phenomenon. The differential diagnosis of thrombocytosis is important, because the clinical course, need of therapy, and prognosis are different in patients with MPDs and in those with reactive thrombocytosis. ET patients may remain asymptomatic for years, but serious thrombohaemorrhagic and pregnancy-related complications may occur. The complications are difficult to predict. The aims of the present study were to evaluate the diagnostic findings, clinical course, and prognostic factors of ET. The present retrospective study consists of 170 ET patients. Two thirds had a platelet count < 1000 x 109/l. The diagnosis was supported by an increased number of megakaryocytes with an abnormal morphology in a bone marrow aspirate, aggregation defects in platelet function studies, and the presence of spontaneous erythroid and/or megakaryocytic colony formation in in vitro cultures of haematopoietic progenitors. About 70 % of the patients had spontaneous colony formation, while about 30 % had a normal growth pattern. Only a fifth of the patients remained asymptomatic. Half had a major thrombohaemorrhagic complication. The proportion of the patients suffering from thrombosis was as high as 45 %. About a fifth had major bleedings. Half of the patients had microvascular symptoms. Age over 60 years increased the risk of major bleedings, but the occurrence of thrombotic complications was similar in all age groups. Male gender, smoking in female patients, the presence of any spontaneous colony formation, and the presence of spontaneous megakaryocytic colony formation in younger patients were identified as risk factors for thrombosis. Pregnant ET patients had an increased risk of complications. Forty-five per cent of the pregnancies were complicated and 38 % of them ended in stillbirth. Treatment with acetylsalicylic acid alone or in combination with platelet lowering drugs improved the outcome of the pregnancy. The present findings about risk factors in ET as well as treatment outcome in the pregnancies of ET patients should be taken into account when planning treatment strategies for Finnish patients.

Aggregation of apolar peptides in organic solvents. Concentration dependence of 1H-nmr parameters for peptide NH groups in 310 helical decapeptide fragment of suzukacillin

Peptide NH chemical shifts and their temperature dependences have been monitored as a function of concentration for the decapeptide, Boc-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-OMe in CDCl3 (0.001-0.06M) and (CD3)2SO (0.001-0.03M). The chemical shifts and temperature coefficients for all nine NH groups show no significant concentration dependence in (CD3)2SO. Seven NH groups yield low values of temperature coefficients over the entire range, while one yields an intermediate value. In CDCl3, the Aib(1) NH group shows a large concentration dependence of both chemical shift and temperature coefficient, in contrast to the other eight NH groups. The data suggest that in (CD3)2SO, the peptide adopts a 310 helical conformation and is monomeric over the entire concentration range. In CDCl3, the 310 helical peptide associates at a concentration of 0.01M, with the Aib(1) NH involved in an intermolecular hydrogen bond. Association does not disrupt the intramolecular hydrogen-bonding pattern in the decapeptide.

Microsphere Bouquets of Bismuth Telluride Nanoplates: Room-Temperature Synthesis and Thermoelectric Properties

We report the synthesis and properties of sphere-shaped microscale aggregates of bismuth telluride nanoplates. We obtain porous microspheres by reducing bismuth chloride and orthotelluric acid with hydrazine in the presence of thioglycolic acid-which serves as the shape-and size-directing agent-followed by room-temperature aging-which promotes nanoplate aggregation. Thin film assemblies of the nanoplate microspheres exhibit n-type behavior due to sulfur doping and a Seebeck coefficient higher than that reported for assemblies of chalcogenide nanostructures. Adaptation of our scalable approach to synthesize and hierarchically assemble nanostructures with controlled doping could be attractive for tailoring novel thermoelectric materials for applications in high-efficiency refrigeration and harvesting electricity from heat.

Aqueous colloidal stability evaluated by zeta potential measurement and resultant TiO2 for superior photovoltaic performance

Controlling the morphological structure of titanium dioxide (TiO 2) is crucial for obtaining superior power conversion efficiency for dye-sensitized solar cells. Although the sol-gel-based process has been developed for this purpose, there has been limited success in resisting the aggregation of nanostructured TiO2, which could act as an obstacle for mass production. Herein, we report a simple approach to improve the efficiency of dye-sensitized solar cells (DSSC) by controlling the degree of aggregation and particle surface charge through zeta potential analysis. We found that different aqueous colloidal conditions, i.e., potential of hydrogen (pH), water/titanium alkoxide (titanium isopropoxide) ratio, and surface charge, obviously led to different particle sizes in the range of 10-500 nm. We have also shown that particles prepared under acidic conditions are more effective for DSSC application regarding the modification of surface charges to improve dye loading and electron injection rate properties. Power conversion efficiency of 6.54%, open-circuit voltage of 0.73 V, short-circuit current density of 15.32 mA/cm2, and fill factor of 0.73 were obtained using anatase TiO 2 optimized to 10-20 nm in size, as well as by the use of a compact TiO2 blocking layer.

Individual variation in in vitro efficacy of antiplatelet medication - aspirin and clopidogrel

Antiplatelet medication is known to decrease adverse effects in patients with atherothrombotic disease. However, despite ongoing antiplatelet medication considerable number of patients suffer from atherothrombotic events. The aims of the study were 1) to evaluate the individual variability in platelet functions and compare the usability of different methods in detecting it, 2) to assess variability in efficacy of antiplatelet medication with aspirin (acetylsalicylic acid) or the combination of aspirin and clopidogrel and 3) to investigate the main genetic and clinical variables as well as potential underlying mechanisms of variability in efficacy of antiplatelet medication. In comparisons of different platelet function tests in 19 healthy individuals PFA-100® correlated with traditional methods of measuring platelet function and was thus considered appropriate for testing individual variability in platelet activity. Efficacy of ongoing 100mg aspirin daily was studied in 101 patients with coronary artery disease (CAD). Aspirin response was measured with arachidonic acid (AA)-induced platelet aggregation, which reflects cyclo-oxygenase (COX)-1 dependent thromboxane (Tx) A2 formation, and PFA-100®, which evaluates platelet activation under high shear stress in the presence of collagen and epinephrine. Five percent of patients failed to show inhibition of AA-aggregation and 21% of patients had normal PFA-100® results despite aspirin and were thus considered non-responders to aspirin. Interestingly, the two methods of assessing aspirin efficacy, platelet aggregation and PFA-100®, detected different populations as being aspirin non-responders. It could be postulated that PFA-100® actually measures enhanced platelet function, which is not directly associated with TxA2 inhibition exerted by aspirin. Clopidogrel efficacy was assessed in 50 patients who received a 300mg loading dose of clopidogrel 2.5 h prior to percutaneous coronary intervention (PCI) and in 51 patients who were given a loading dose of 300mg combined with a five day treatment of 75mg clopidogrel daily mimicking ongoing treatment. Clopidogrel response was assessed with ADP-induced aggregations, due to its mechanism of action as an inhibitor of ADP-induced activation. When patients received only a loading dose of clopidogrel prior to PCI, 40% did not gain measurable inhibition of their ADP-induced platelet activity (inhibition of 10% or less). Prolongation of treatment so that all patients had reached a plateau of inhibition exerted by clopidogrel, decreased the incidence of non-responders to 20%. Polymorphisms of COX-1 and GP VI, as well as diabetes and female gender, were associated with decreased in vitro aspirin efficacy. Diabetes also impaired the in vitro efficacy of short-term clopidogrel. Decreased response to clopidogrel was associated with limited inhibition by ARMX, an antagonist of P2Y12-receptor, suggesting the reason for clopidogrel resistance to be receptor-dependent. Conclusions: Considerable numbers of CAD patients were non-responders either to aspirin, clopidogrel or both. In the future, platelet function tests may be helpful to individually select effective and safe antiplatelet medication for these patients.

Design, Synthesis, and DNA Binding Properties of Photoisomerizable Azobenzene−Distamycin Conjugates: An Experimental and Computational Study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene−distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.

Design of mechanism-based inhibitors of transthyretin amyloidosis:Studies with biphenyl ethers and new structural templates

Transthyretin (TTR), a tetrameric thyroxine (T4) carrier protein, is associated with a variety of amyloid diseases. In this study, we explore the potential of biphenyl ethers (BPE), which are shown to interact with a high affinity to its T4 binding site thereby preventing its aggregation and fibrillogenesis. They prevent fibrillogenesis by stabilizing the tetrameric ground state of transthyretin. Additionally, we identify two new structural templates (2-(5-mercapto-[1,3,4]oxadiazol-2-yl)-phenol and 2,3,6-trichloro-N-(4H-[1,2,4]triazol-3-yl) represented as compounds 11 and 12, respectively, throughout the manuscript) exhibiting the ability to arrest TTR amyloidosis. The dissociation constants for the binding of BPEs and compound 11 and 12 to TTR correlate with their efficacies of inhibiting amyloidosis. They also have the ability to inhibit the elongation of intermediate fibrils as well as show nearly complete (> 90%) disruption of the preformed fibrils. The present study thus establishes biphenyl ethers and compounds 11 and 12 as very potent inhibitors of TTR fibrillization and inducible cytotoxicity.

An investigation into the thermophysical and rheological properties of nanofluids for solar thermal applications

Considered to be the next generation of heat transfer fluids, nanofluids have been receiving a growing amount of attention in the past decade despite the controversy and inconsistencies that have been reported. Nanofluids have great potential in a wide range of fields, particularly for solar thermal applications. This paper presents a comprehensive review of the literature on the enhancements in thermophysical and rheological properties resulting from experimental works conducted on molten salt nanofluids that are used in solar thermal energy systems. It was found that an increase in specific heat of 10–30% was achieved for most nanofluids and appeared independent of particle size and to an extent mass concentration. The specific heat increase was attributed to the formation of nanostructures at the solid–liquid interface and it was also noted that the aggregation of nanoparticles has detrimental effects on the specific heat increase. Thermal conductivity was also found to increase, though less consistently, ranging from 3% to 35%. Viscosity was seen to increase with the addition of nanoparticles and is dependent on the amount of aggregation of the particles. An in-depth micro level analysis of the mechanisms behind the thermophysical property changes is presented in this paper. In addition, possible trends are discussed relating to current theorised mechanisms in an attempt to explain the behaviour of molten salt nanofluids.

Electric Field–Enhanced Sensitivity of Grafted Ligands and Receptors

BACKGROUND: Particle-based agglutination tests consisting of receptors grafted to colloidal microparticles are useful for detecting small quantities of corresponding ligands of interest in fluid test samples, but detection limits of such tests are limited to a certain concentration and it is most desirable to lower the detection limits and to enhance the rate of recognition of ligands. METHODS: A mixture of receptor-coated colloidal microparticles and corresponding ligand was sandwiched between 2 indium tin oxide-coated glass plates. Electrohydrodynamic drag from an alternating-current electric field applied perpendicular to the plates increased the local concentration of the colloidal particles, improving the chances of ligand-receptor interaction and leading to the aggregation of the colloidal particles. RESULTS: With this technique the sensitivity of the ligand-receptor recognition was increased by a factor as large as 50. CONCLUSIONS: This method can improve the sensitivity of particle-based agglutination tests used in immuno-assays and many other applications such as immunoprecipitation and chemical, sniffing. (C) 2007 American Association for Clinical Chemistry.

Visualization of in situ intracellular aggregation of two cataract-associated human gamma-crystallin mutants: Lose a tail, lose transparency

PURPOSE. To understand the molecular features underlying autosomal dominant congenital cataracts caused by the deletion mutations W156X in human gamma D-crystallin and W157X in human gamma C-crystallin. METHODS. Normal and mutant cDNAs (with the enhanced green fluorescent protein [EGFP] tag in the front) were cloned into the pEGFP-C1 vector, transfected into various cell lines, and observed under a confocal microscope for EGFP fluorescence. Normal and W156X gamma D cDNAs were also cloned into the pET21a(+) vector, and the recombinant proteins were overexpressed in the BL-21(DE3) pLysS strain of Escherichia coli, purified, and isolated. The conformational features, structural stability, and solubility in aqueous solution of the mutant protein were compared with those of the wild type using spectroscopic methods. Comparative molecular modeling was performed to provide additional structural information. RESULTS. Transfection of the EGFP-tagged mutant cDNAs into several cell lines led to the visualization of aggregates, whereas that of wild-type cDNAs did not. Turning to the properties of the expressed proteins, the mutant molecules show remarkable reduction in solubility. They also seem to have a greater degree of surface hydrophobicity than the wild-type molecules, most likely accounting for self-aggregation. Molecular modeling studies support these features. CONCLUSIONS. The deletion of C-terminal 18 residues of human gamma C-and gamma D-crystallins exposes the side chains of several hydrophobic residues in the sequence to the solvent, causing the molecule to self-aggregate. This feature appears to be reflected in situ on the introduction of the mutants in human lens epithelial cells.