A hybrid molecule that prohibits amyloid fibrils and alleviates neuronal toxicity induced by beta-amyloid (1-42)

Inhibition of oligomeric amyloid beta (Abeta) peptide or fibril formation has emerged as a major therapeutic target for developing new drugs for Alzheimer's disease. We focused on developing inhibitors by synthesizing hybrid molecules of ferulic acid and styryl benzene, which has been known as a fibril binder. Initially, cell-based assay was carried out to evaluate the effective compound. A selected effector, 1, alleviated the Abeta-induced neuronal toxicity in differentiated SH-SY5Y human neuroblastoma cells. The effector could also inhibit Abeta fibril formation, monitored by thioflavin T fluorescence intensity assay and transmitted electron microscopic images. A strong binding affinity of 1 to non-fibrous monomer-like Abeta, which was immobilized to a surface chip, was measured using a surface plasmon resonance technique. The data suggest that the effector shifts the equilibrium of multimeric Abeta, inhibiting the pathogenic oligomer or fibril formation.     

Ferulic acid ethyl ester protects neurons against amyloid beta- peptide(1-42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity

Alzheimer's disease (AD) is neuropathologically characterized by depositions of extracellular amyloid and intracellular neurofibrillary tangles, associated with loss of neurons in the brain. Amyloid beta-peptide (Abeta) is the major component of senile plaques and is considered to have a causal role in the development and progress of AD. Several lines of evidence suggest that enhanced oxidative stress and inflammation play important roles in the pathogenesis or progression of AD. The present study aimed to investigate the protective effects of ethyl-4-hydroxy-3-methoxycinnamic acid (FAEE), a phenolic compound which shows antioxidant and anti-inflammatory activity, on Abeta(1-42)-induced oxidative stress and neurotoxicity. We hypothesized that the structure of FAEE would facilitate radical scavenging and may induce protective proteins. Abeta(1-42) decreases cell viability, which was correlated with increased free radical formation, protein oxidation (protein carbonyl, 3-nitrotyrosine), lipid peroxidation (4-hydroxy-2-trans-nonenal) and inducible nitric oxide synthase. Pre-treatment of primary hippocampal cultures with FAEE significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular reactive oxygen species accumulation, protein oxidation, lipid peroxidation and induction of inducible nitric oxide synthase. Treatment of neurons with Abeta(1-42) increases levels of heme oxygenase-1 and heat shock protein 72. Consistent with a cellular stress response to the Abeta(1-42)-induced oxidative stress, FAEE treatment increases the levels of heme oxygenase-1 and heat shock protein 72, which may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells against Abeta(1-42)-induced toxicity. These results suggest that FAEE exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress directly and by inducing protective genes. These findings suggest that FAEE could potentially be of importance for the treatment of AD and other oxidative stress-related diseases.     

The effects of simultaneous variation in protein, digestible carbohydrate and tannic acid on the feeding behaviour of larval Locusta migratoria (L.) and Schistocerca gregaria (Forskal). I. Short-term studies

ABSTRACT The influence of simultaneously varying the levels in artificial diets of protein, digestible carbohydrate (14% or 28%) and tannic acid (absent or 10%) on the feeding behaviour of the oligophagous Locusta migratoria (L.) and the polyphagous Schistocerca gregaria (Forskal) (Acrididae) was investigated. Total consumption and detailed feeding behaviour were recorded over a 12 h period in choice and no-choice experiments. In addition, amounts eaten by Schistocerca of the 14% protein, 14% carbohydrate diet with and without tannic acid were measured at regular intervals throughout the fifth stadium, and insect growth over this period was recorded. There were no interactive effects of nutrient levels and tannic acid, despite the fact that both species compensated for dilution of dietary protein by increasing consumption. Only male Locusta compensated for dilution of dietary carbohydrates, and this compensation was much less marked than for protein. Tannic acid did influence feeding as a main effect, however. It caused an increase in amounts eaten by Schistocerca in both choice and no-choice experiments. This increased consumption was due to an increase in the number of meals taken. A shorter latency period before and a longer duration of the first meal by naive insects suggested a phagostimulatory rather than a post-ingestive effect of tannic acid. The stimulatory effect was only apparent for the first 24 h of continuous exposure, but this temporary enhancement none the less resulted in the insects being heavier at adult ecdysis. Stadium duration was also somewhat reduced. In a no-choice situation, no effect of tannic acid on the feeding behaviour of Locusta was observed. When given a choice, however, this species took significantly more meals on the tannic acid-free diet, these being of similar average size to meals taken on the tannic acid diet. Significantly more insects took their first meals on the tannic acid-free diet in the choice test, indicating a deterrent effect of tannic acid in Locusta.     

Kinetics of c-reactive protein (CRP) and serum amyloid A protein (SAA) in patients with community-acquired pneumonia (CAP), as presented with biologic half-life times

Context: In management of community-acquired pneumonia (CAP), excellent biomarkers for inflammation would be helpful in our practice.

Objectives: Kinetics of c-reactive protein (CRP) and serum amyloid A (SAA) was characterized, using their biologic half-life times.

Materials and methods: Time course of CRP and SAA levels in the successfully treated 36 CAP patients were investigated and their half-life times were determined and compared.

Results & Discussions: SAA and CRP declined in an exponential mean and the biologic half-life times of SAA levels was 34.9?±?28.7?h, significantly shorter than that of CRP, 46.4?±?21.7?h (p?=?0.0014). Conclusion: The kinetic evidence, presented as biologic half-life times of CRP and SAA, helps us make a clinical assessment of CAP patients.     

Biochemical and morphological changes in carp (Cyprinus carpio L.) liver following exposure to copper sulfate and tannic acid

As a consequence of human activity various toxicants reach the aquatic ecosystems; humics may interact with them and may change their toxicity. Many fish are exposed to a considerable concentration of humics and pollutants. Because of paucity of data on the biochemical action of tannins in the presence of the fungicide CuSO4 a comparative study was undertaken. The alterations of redox-parameters in carp liver were monitored and tissue necrosis was followed by measuring the plasma transaminase activities and by electron microscopy. Tannic acid, a representative phenolic/humic compound, exerted prooxidant effects in carp, which may be partially due to formation of prooxidant intermediates/end-products via its biotransformation. Alternatively, tannic acid may partially inhibit the antioxidant enzymes of fish. The response to CuSO4 was more severe. Although tannic acid alone acted as a prooxidant in fish, electron micrographs demonstrated that it reduced the necrotizing effect of copper, which may be due to the complexing activity of tannic acid with the biomolecules of the hepatocytes and to the H2O2-degrading activity of tannin-CuSO4 combination. Our results indicate that the heavy metal-detoxifying capacity of tannin may be significant; however, tannin-exposure alone or combined with metals may be toxic for fish due to enzyme inhibition and oxidative stress induction.     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Comparison between intact and desialylated human serum amyloid P component by laser photo CIDNP (chemically induced dynamic nuclear polarization) technique: an indication for a conformational impact of sialic acid

The human pentraxin serum amyloid P component (SAP) exhibits no microheterogeneity in its complex di-antennary glycan. To elucidate whether the removal of sialic acids from this glycoprotein might affect the accessibility of certain amino acid residues of the protein we employed the laser photo CIDNP approach as a sensitive tool. The CIDNP effect is generated by the interaction of a photoexcited dye with reactive amino acids and results in enhanced absorption- or emission-signals which can be observed for the three aromatic amino acids histidine, tryptophan, and tyrosine if they are accessible to the dye. Therefore, this technique can be applied to explore surface exposure of these amino acid residues. The respective spectra of SAP and enzymatically desialylated SAP were determined. Six tryptophan/histidine signals and one tyrosine signal are present in the aromatic part of the CIDNP difference spectrum of SAP. The corresponding spectrum of desialylated SAP shows remarkable alterations. The chemical shift of one Trp/His-characteristic signal is decreased by 0.1 ppm. One Trp/His-signal disappeared and a new one was formed in the CIDNP difference spectrum of desialylated SAP, while the other signals were unaffected. The Tyr signal has a clearly enhanced intensity in desialylated SAP. Therefore, the removal of sialic acid moieties from the single N-glycan of each monomer apparently affects surface presentation of distinct CIDNP-reactive amino acids of SAP [1]. A conformational change of the protein part of SAP in relation with a different orientation of the desialylated oligosaccharide chain in comparison to the complete one is a possible explanation of our CIDNP results. This revised version was published online in August 2006 with corrections to the Cover Date.     

The release of glutamate and aspartate from rat brain synaptosomes in response to domoic acid (amnesic shellfish toxin) and kainic acid

Kainic acid is known to stimulate the release of glutamate (GLU) and aspartate (ASP) from presynaptic neurons. It has been suggested that the enhanced release of these endogenous EAA's plays a significant role in the excitotoxic effects of KA. Domoic acid (DOM), a shellfish toxin, is structurally similar to KA, and has been shown to be 3–8 times more toxic than KA. In this study, effects of KA and DOM on the release of GLU and ASP from rat brain synaptosomes were investigated. Amino acid analysis was performed by the reversed phase HPLC, following derivatization with 9-fluorenylmethyl chloroformate (FMOC). Potassium chloride (40 mM) was used as a positive control, and stimulated GLU release from rat brain synaptosomes in presence or absence of Ca²⁺. DOM enhanced the release of GLU, whereas KA stimulated the release of both GLU and ASP from synaptosomes in the presence of Ca²⁺. However, their potency to stimulate GLU and ASP release was enhanced in absence of Ca²⁺. These results indicate that diferent mechanisms may be involved in the release of GLU and ASP in response to DOM and KA, and that neurotransmitter release appeared to be highly specific for these agonists. It would appear that DOM and KA may interact with different receptors on the presynaptic nerve terminal, and/or activate different subtypes of voltage-dependent Ca²⁺ channels to promote influx of Ca²⁺ which is targeted for different pools of neurotransmitters.Abbreviations ANOVA analysis of variance - ASP aspartate - DOM domoic acid - DHKA dihydrokainic acid - EAA excitatory amino acid - FMOC 9-fluorenylmethyl chloroformate - GLU glutamate - KA kainic acid     

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full-length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1-eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP+1-eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1-eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.     

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.     

Flotation separation of mercury(II) from environmental water samples using thiosemicarbazide derivatives as chelating agents and oleic acid as surfactant

Abstract

A simple and rapid procedure is developed for the quantitative flotation of mercury(II) from aqueous solutions. Thiosemicarbazide derivatives such as: 1-(amino-N-phenylmethanethio)-4-(pyridine-2-yl)thio-semicarbazide (H₂PPS), N-phenyl-2-(pyridine-2-ylcarbamothioyl) hydrazinecarboxamide (H₂PBO), 1-(amino(thioformyl)-N-phenylform)-4-(pyridine-2-yl)thiosemicarbazide (H2APO), and 1-(amino-N-(pyri-dine-3-yl)methanethio)-4-(pyridine-2-yl)thiosemicarbazide (H₂PPY) are used as organic chelating agents and oleic acid (HOL) as surfactant. The different parameters affecting the flotation process namely, metal ion, ligands and surfactant concentrations, foreign ions (which are normally present in fresh and saline waters), pH and temperature are examined. Nearly 100% of mercury ions are floated at a metal:ligand ratio of 1: 4, pH ~ 4 and at room temperature (~25°C). The procedure was successfully applied to recover Hg²⁺ ions spiked into some water samples. A flotation mechanism is suggested based on some physical and chemical studies on the solid complexes isolated from aqueous and floated layers.     

A monoclonal antibody to (S)-abscisic acid: its characterisation and use in a radioimmunoassay for measuring abscisic acid in crude extracts of cereal and lupin leaves

A monoclonal antibody produced to abscisic acid (ABA) has been characterised and the development of a radioimmunoassay (RIA) for ABA using the antibody is described. The antibody had a high selectivity for the free acid of (S)-cis, trans-ABA. Using the antibody, ABA could be assayed reliably in the RIA over a range from 100 to 4000 pg (0.4 to 15 pmol) ABA per assay vial. As methanol and acetone affected ABA-antibody binding, water was used to extract ABA from leaves. Water was as effective as aqueous methanol and acetone in extracting the ABA present. Crude aqueous extracts of wheat, maize and lupin leaves could be analysed without serious interference from other immunoreactive material. This was shown by measuring the distribution of immunoreactivity in crude extracts separated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), or by comparing the assay with physicochemical methods of analysis. Analysis of crude extracts by RIA and either, after TLC purification, by gas chromatography using an electron-capture detector or, after HPLC purification, by combined gas chromatography-mass spectrometry (GC-MS) gave very similar ABA concentrations in the initial leaf samples. However, RIA analysis of crude aqueous extracts of pea seeds resulted in considerable overestimation of the amount of ABA present. Determinations of ABA content by GC-MS and RIA were similar after pea seed extracts had been purified by HPLC. Although the RIA could not be used to analyse ABA in crude extracts of pea seeds, it is likely that crude extracts of leaves of several other species may be assayed successfully.Abbreviations ABA abscisic acid - DW dry weight - FW fresh weight - GC-ECD gas chromatography using an electron capture detector - GC-MS combined gas chromatographymass spectrometry - HPLC high-performance liquid chromatography - McAb monoclonal antibody - PVP soluble polyvinylpyrrolidone - RIA radioimmunoassay - TLC thin-layer chromatography     

Pseudo-nitzschia and domoic acid fluxes in Santa Barbara Basin (CA) from 1993 to 2008

Blooms of domoic acid (DA) producing Pseudo-nitzschia, regularly occur off the coast of California. Although it has been hypothesized that these blooms are increasing in frequency, the lack of historical records limits our understanding of potential causal mechanisms. In this study, an 15-year time-series (1993–2008) of sediment trap samples collected from the Santa Barbara Basin (SBB) at 540 m were analyzed for Pseudo-nitzschia (n = 196, microscopy and SEM) and DA (n = 206, LC–MS/MS) concentrations and fluxes. Results suggest that there was an abrupt shift towards greater frequency and higher magnitude Pseudo-nitzschia blooms and toxic DA flux events in the SBB after the year 2000. SEM analysis of sediment trap material indicates that these events were mainly blooms of P. australis, with cell fluxes increasing by an order of magnitude from a maximum of 4.5 × 10⁶ cells m⁻² d⁻¹ pre-2000, to as high as 3.2 × 10⁸ cells m⁻² d⁻¹ thereafter. Similarly, sediment trap DA fluxes increased by an average of 13.4 μg m⁻² d⁻¹, with only one large event (>5 μg m⁻² d⁻¹) from 1993 to 1999 versus 16 large DA events from 2000 to 2008. While the causes of this abrupt shift remain ambiguous, we suggest that this shift may be related to natural climate variability associated with a change in phase of the North Pacific Gyre Oscillation (NPGO) and its potential influence on the composition and magnitude of waters that are upwelled into the SBB.     

Ascorbic acid and flavonoid-peroxidase reaction as a detoxifying system of H(2)O(2) in grapevine leaves

Biosynthesis of both ascorbic acid (AsA) and peroxidase activity were induced by light in cv. Sultana grapevine leaves. Induced peroxidase activity mainly involved basic isoenzymes of pI 9.8 and 9.6 and catalyzed the oxidation of flavonoids like quercetin and kaempferol and derivatives of hydroxycinnamic acids such as ferulic and p-coumaric acids, but not AsA. However, the peroxidase-dependent oxidation of ferulic acid and quercetin was temporarily suppressed by AsA as long as it remained in the reaction medium. Kinetics and spectroscopic results indicated that AsA was oxidized to dehydroascorbic acid only in the presence of phenols or flavonoids, and did not interfere with the catalytic activity of the peroxidase. Ascorbate peroxidase isoenzymes (APx), whose activities are widely considered central for detoxification of H(2)O(2) in most plant cells, were not detected in grape leaves extracts. The significance of light stimulus on peroxidase activity and leaf AsA content is discussed in terms of a flavonoid-redox cycle proposed as an alternative system to detoxify H(2)O(2) in grapevine leaves.     

Catabolism of indole-3-acetic acid to indole-3-methanol in a crude enzyme extract and in protoplasts from Scots pine (Pinus sylvestris)

The antagonistic effects of ethylene and Ag⁺ on the metabolism of [1-¹⁴C]indole-3-acetic acid (IAA) and on the rates of ethylene production were studied in tobacco leaf discs ( Nicotiana rustica var. Brasilia ). During the first 10 h of incubation, Ag⁺-pretreated leaf discs contained more free [¹⁴C]IAA than untreated ones due to decreased oxidative decarboxylation, and the discs also produced more ethylene. Exogenously supplied ethylene nullified these effects of Ag⁺. However, the most pronounced effect of Ag⁺ in increasing ethylene production, as well as the strongest antagonistic effect of exogenous ethylene, were found between 24 and 48 h of incubation. During this time span no effect on the level of free IAA and on its decarboxylation could be observed. It is suggested that ethylene exerted its autoinhibitory effect by a feedback control on the IAA-induced ethylene biosynthesis. Possible mechanisms for the autoinhibitory effect of ethylene are discussed.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-¹⁴C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [¹⁴C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-¹⁴C]20:5n-3 to [3-¹⁴C]22:6n-3 was more efficient than that of [1-¹⁴C]20:4n-6 to [3-¹⁴C]22:5n-6. At low substrate concentration (4 μM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 μM). The conversion of [1-¹⁴C]22:5n-3 to [1-¹⁴C]22:6n-3 was 1.7 times more efficient than that of [1-¹⁴C]22:4n-6 to [1-¹⁴C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-¹⁴C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-¹⁴C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-¹⁴C]20:4n-6 or [1-¹⁴C]22:4n-6 to [¹⁴C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-¹⁴C]20:5n-3 and [1-¹⁴C]22:5n-3 to [¹⁴C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.