Strictinin as an efficient antioxidant in lipid peroxidation

The antioxidant effect of strictinin (SOH), which was extracted from green tea leaves, against the peroxidation of linoleic acid in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium (CTAB) micelles, against the peroxidation of low-density lipoprotein (LDL) and against oxidative hemolysis of human red blood cells (RBCs), has been studied. The peroxidation of linoleic acid and LDL, and oxidative hemolysis of RBCs were initiated thermally by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), and the reaction kinetics in micelles and LDL were monitored by uptake of oxygen. The synergistic antioxidant effect of SOH with alpha-tocopherol (Vitamin E) was also studied by following the decay kinetics of alpha-tocopherol. Kinetic analysis of the antioxidation process demonstrates that SOH, used either alone or in combination with alpha-tocopherol, is an effective antioxidant against lipid peroxidation, but its effects significantly depend on the reaction medium.     

Moderate exercise is an antioxidant: upregulation of antioxidant genes by training

Exercise causes oxidative stress only when exhaustive. Strenuous exercise causes oxidation of glutathione, release of cytosolic enzymes, and other signs of cell damage. However, there is increasing evidence that reactive oxygen species (ROS) not only are toxic but also play an important role in cell signaling and in the regulation of gene expression. Xanthine oxidase is involved in the generation of superoxide associated with exhaustive exercise. Allopurinol (an inhibitor of this enzyme) prevents muscle damage after exhaustive exercise, but also modifies cell signaling pathways associated with both moderate and exhaustive exercise in rats and humans. In gastrocnemius muscle from rats, exercise caused an activation of MAP kinases. This in turn activated the NF-kappaB pathway and consequently the expression of important enzymes associated with defense against ROS (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when ROS production was prevented by allopurinol. Thus ROS act as signals in exercise because decreasing their formation prevents activation of important signaling pathways that cause useful adaptations in cells. Because these signals result in an upregulation of powerful antioxidant enzymes, exercise itself can be considered an antioxidant. We have found that interfering with free radical metabolism with antioxidants may hamper useful adaptations to training.     

Escherichia coli flavohemoglobin is an efficient alkylhydroperoxide reductase

Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low Km toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical peroxidases, HMP turns over in the ferrous state. In particular, an iron oxygen adduct intermediate whose spectrum is similar to that reported for the oxo-ferryl derivative in peroxidases (Compound II), has been identified during the catalysis of hydrogen peroxide reduction. This finding suggests that hydroperoxide cleavage occurs upon direct binding of a peroxide oxygen atom to the ferrous heme iron. Competitive inhibition of the alkylhydroperoxide reductase activity by carbon monoxide has also been observed, thus confirming that heme iron is directly involved in the catalytic mechanism of hydroperoxide reduction. The alkylhydroperoxide reductase activity taken together with the unique lipid binding properties of HMP suggests that this protein is most likely involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.     

The antihypertensive hydralazine is an efficient scavenger of acrolein

Recent work indicates the highly toxic alpha,beta-unsaturated aldehyde acrolein is formed during the peroxidation of polyunsaturated lipids, raising the possibility that it functions as a 'toxicological second messenger' during oxidative cell injury. Acrolein reacts rapidly with proteins, forming adducts that retain carbonyl groups. Damage by this route may thus contribute to the burden of carbonylated proteins in tissues. This work evaluated several amine compounds with known aldehyde-scavenging properties for their ability to attenuate protein carbonylation by acrolein. The compounds tested were: (i) the glycoxidation inhibitors, aminoguanidine and carnosine; (ii) the antihypertensive, hydralazine; and (iii) the classic carbonyl reagent, methoxyamine. Each compound attenuated carbonylation of a model protein, bovine serum albumin, during reactions with acrolein at neutral pH and 37 degrees C. However, the most efficient agent was hydralazine, which strongly suppressed carbonylation under these conditions. Study of the rate of reaction between acrolein and the various amines in a protein-free buffered system buttressed these findings, since hydralazine reacted with acrolein at rates 2-3 times faster than its reaction with the other scavengers. Hydralazine also protected isolated mouse hepatocytes against cell killing by allyl alcohol, which undergoes in situ alcohol dehydrogenase-catalysed conversion to acrolein.     

Fibrinogen is chemotactic for vascular smooth muscle cells

We studied the effect of fibrinogen on the migration of bovine aortic smooth muscle cells in culture, using a Neuro Probe 48-well micro chemotaxis chamber. Fibrinogen stimulated the migration of the cells dose-dependently at concentrations from 30 to 1000 micrograms/ml. A modified checkerboard analysis of the response demonstrated that the effect was largely chemotactic in nature. The present results suggest that fibrinogen may play an important role in the pathogenesis of arterial intimal thickening and atherosclerosis.     

D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae

D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8kb in size. In the alo1 mutants, D-erythroascorbic acid and the activity of D-arabinono-1,4-lactone oxidase could not be detected. The intracellular concentration of D-erythroascorbic acid and the enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.     

Sulfide is an efficient iron releasing agent for mammalian ferritins

The most prominent role of mammalian ferritins is to provide an extensive iron-buffering capacity to cells. The large ferritin iron stores can be mobilized in vitro, but the functional relevance of the most efficient iron releasing agents remains elusive. Sulfide is a strongly reducing chemical generated by a series of enzymes. In the presence of limited amounts of sulfide a continuous rate of iron release from ferritin was observed and a majority of the protein iron core was recovered in solution. The rate constants for iron efflux triggered by several reducing or chelating compounds have been measured and compared. Although not as efficient as reduced flavins, sulfide displayed kinetic parameters which suggest a potential physiological role for the chalcogenide in converting the iron storage protein into apoferritin. To further probe the relevance of sulfide in the mobilization of iron, several enzymes, such as NifS, rhodanese, or sulfite reductase generating reduced forms of sulfur by different mechanisms, have been assayed for their ability to catalyze the release of iron from ferritin. The results show that full reduction of sulfur into sulfide is needed to deplete iron from ferritin. These reactions suggest links between sulfur metabolism and intracellular iron homeostasis.     

Low molecular weight chitosan is an efficient inhibitor of ribonucleases

RNase inhibitors are commonly used to block the RNase activity in manipulations with RNA-containing preparations. Recently RNase inhibitors, either synthetic or natural, have been intensively sought because they appeared to be promising for therapy of cancer and allergy. However, there is only a limited number of efficient RNase inhibitors. We have shown that a low molecular weight chitosan (M(r) approximately 6 kDa) inhibits activity of pancreatic RNase A and some bacterial RNases with inhibition constants in the range of 30-220 nM at pH 7.0 and ionic strength 0.14 M. The preferential contribution to the chitosan complex formation with RNases is due to establishment of 5-6 ion pairs. The results of this work show that polycations may efficiently inhibit ribonuclease activities.     

Fibrinogen beta-chain tyrosine nitration is a prothrombotic risk factor

Elevated levels of circulating fibrinogen are associated with an increased risk of atherothrombotic diseases although a causative correlation between high levels of fibrinogen and cardiovascular complications has not been established. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Mass spectrometry identified tyrosine residues 292 and 422 at the carboxyl terminus of the beta-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob "B" mimetic peptides Gly-His-Arg-Pro(am) and Ala-His-Arg-Pro(am) suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.     

Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae

Abstract The tripeptide γ-l-glutamyl-l-cystinylglycine (glutathione) is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. We wished to determine the role of glutathione in the protection of the yeast Saccharomyces cerevisiae against oxidative stress. This study shows that glutathione is an important antioxidant molecule in yeast, with γ-glutamylcysteine synthetase ( gshI ) mutants, deficient in glutathione synthesis, being hypersensitive to H₂O₂ and Superoxide anions in both exponential- and stationary-phase cultures. Despite this, these mutants are still able to induce adaptive stress responses to oxidants.     

Amyloid-beta is an antioxidant for lipoproteins in cerebrospinal fluid and plasma

Amyloid-beta (Abeta) peptide, a major constituent of senile plaques and a hallmark of Alzheimer's disease (AD), is normally secreted by neurons and can be found in low concentrations in cerebrospinal fluid (CSF) and plasma, where it is associated with lipoproteins. However, the physiological role of Abeta secretion remains unknown. Here we show that at the concentrations measured in biological fluids (0.1-1.0 nM), Abeta(1-40) strongly inhibits autooxidation of CSF lipoproteins and plasma low density lipoprotein (LDL). At higher concentrations of the peptide its antioxidant action was abolished. Abeta(1-40) also inhibited copper-catalyzed LDL oxidation when added in molar excess of copper, but did not influence oxidation induced by an azo-initiator. Other Abeta peptides also possessed antioxidant activity in the order Abeta(1-40) > Abeta(1-42) > Abeta(25-35), whereas Abeta(35-25) was inactive. These data suggest that Abeta(1-40) may act as a physiological antioxidant in CSF and plasma lipoproteins, functioning by chelating transition metal ions.     

Chemically modified firefly luciferase is an efficient source of near-infrared light

Bioluminescence and bioluminescence resonance energy transfer (BRET) are two naturally occurring light emission phenomena that have been adapted to a wide variety of important research applications including in vivo imaging and enzyme assays. The luciferase enzyme from the North American firefly, which produces yellow-green light, is a key component of many of these applications. Recognizing the heightened interest in the potential of near-infrared (nIR) light to improve these technologies, we have demonstrated that spectral emissions with maxima of 705 and 783 nm can be efficiently produced by a firefly luciferase variant covalently labeled with nIR fluorescent dyes. In one case, an outstanding BRET ratio of 34.0 was achieved emphasizing the importance of selective labeling with fluorescent dyes and the efficiency provided by the intramolecular BRET process. Additionally, we constructed a biotinylated fusion protein that similarly produced nIR light. This novel material was immobilized on solid supports containing streptavidin, demonstrating, in principle, that it may be used for receptor-based imaging. Also, the matrix-bound labeled fusion protein was used to measure factor Xa activity at physiological concentrations in blood. We believe this to be the first report of bright nIR light sources produced by chemical modification of a beetle luciferase.     

3-Nitrocoumarin is an efficient inhibitor of budding yeast phospholipase-C.

3-Nitrocoumarin is described in the literature as a specific inhibitor of mammalian phospholipase-C and here we studied the effect of 3-nitrocoumarin on budding yeast phosphatidylinositol-specific phospholipase-C and its effect on yeast growth. 3-Nitrocoumarin is a powerful inhibitor in vitro of the yeast Plc1 protein with an IC(50) of 57 nM and it is also an inhibitor of yeast growth in minimal media at comparable concentrations. Moreover at the same concentration it inhibits the glucose-induced PI-turnover. Since the effects of 3-nitrocoumarin on yeast growth are superimposable on the growth phenotype caused by PLC1 gene deletion we can conclude that 3-nitrocoumarin is a specific and selective inhibitor of yeast phospholipase-C. In addition we show that 3-nitrocoumarin was also an effective inhibitor of the pathogenic yeast Candida albicans.     

The catecholic antioxidant piceatannol is an effective nitrosation inhibitor via an unusual double bond nitration

Piceatannol (1) was found to be more effective than caffeic acid, an established antinitrosating agent, in inhibiting N-nitrosation of 2,3-diaminonaphthalene. Product analysis of the reaction mixture of 1 (20 microM) with nitrite ions (80 microM) at pH 3.0 and at 37 degrees C showed conversion to a single major nitration product, (E)-3,3',4,5'-tetrahydroxy-beta-nitrostilbene (2) (68% yield). This would result from an unexpected nitration at the double bond sector via the 4-phenoxyl radical, which was analyzed at the unrestricted DFT level.     

Nucleolin is required for an efficient herpes simplex virus type 1 infection

Productive infection by herpes simplex virus type 1 (HSV-1), which occurs in the host cell nucleus, is accompanied by dramatic modifications of the nuclear architecture, including profound alterations of nucleolar morphology. Here, we show that the three most abundant nucleolar proteins--nucleolin, B23, and fibrillarin--are redistributed out of the nucleoli as a consequence of HSV-1 infection. We show that the amount of nucleolin increases progressively during the course of infection. We demonstrate for the first time that a nucleolar protein, i.e., nucleolin, colocalizes with ICP8 in the viral replication compartments, at the time when viral replication is effective, suggesting an involvement of nucleolin in the HSV-1 DNA replication process. At later times of infection, a granular form of nucleolin localizes to the cytoplasm, in structures that display the characteristic features of aggresomes, indicating that this form of nucleolin is very probably destined for degradation. The delocalization of nucleolin from the nucleoli requires the viral ICP4 protein or a factor(s) whose expression involves ICP4. Using small interfering RNA technology, we show that viral replication requires a high level of nucleolin expression, demonstrating for the first time a direct role for a nucleolar protein in herpes simplex virus biology.     

MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants

The conjugation of the ubiquitin-like modifier NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN8/RELATED TO UBIQUITIN1 (NEDD8/RUB1; neddylation) is best known as an important posttranslational modification of the cullin subunits of cullin-RING-type E3 ubiquitin ligases (CRLs). MLN4924 has recently been described as an inhibitor of NEDD8-ACTIVATING ENZYME1 (NAE1) in human. Here, we show that MLN4924 is also an effective and specific inhibitor of NAE1 enzymes from Arabidopsis (Arabidopsis thaliana) and other plant species. We found that MLN4924-treated wild-type seedlings have phenotypes that are highly similar to phenotypes of mutants with a partial defect in neddylation and that such neddylation-defective mutants are hypersensitive to MLN4924 treatment. We further found that MLN4924 efficiently blocks the neddylation of cullins in Arabidopsis and that MLN4924 thereby interferes with the degradation of CRL substrates and their downstream responses. MLN4924 treatments also induce characteristic phenotypes in tomato (Solanum lycopersicum), Cardamine hirsuta, and Brachypodium distachyon. Interestingly, MLN4924 also blocks the neddylation of a number of other NEDD8-modified proteins. In summary, we show that MLN4924 is a versatile and specific neddylation inhibitor that will be a useful tool to examine the role of NEDD8- and CRL-dependent processes in a wide range of plant species.     

PON1 paraoxonase activity is reduced during HDL oxidation and is an indicator of HDL antioxidant capacity

The aim of this study was to investigate the effect of HDL oxidation on PON1 paraoxonase activity. Also, we were interested in investigating the mechanism by which PON1 could be inactivated and the correlation between its enzymatic activity and the antioxidant properties of HDL. Three different oxidation systems were used for the HDL oxidation: (1) oxidation induced by THP1 cells, (2) oxidation induced by copper ions at a concentration 10 microM, and (3) oxidation induced by *OH and O2.- oxygen free radicals produced by gamma-radiolysis. HDL oxidation was followed by the measurement of lipid peroxide formation, and PON1 activity was determined by measuring the rate of paraoxon hydrolysis. Our results show that HDL oxidation is accompanied by a reduction in the PON1 paraoxonase activity. The extent of PON1 inactivation depends both on the extent of HDL oxidation and on the oxidation system used. The rates of HDL oxidation and PON1 inactivation were significantly correlated (r = 0.93, p < 0.0054). Our results show that oxidized HDL loses its protective effect toward LDL oxidation. The antioxidant action of HDL towards LDL oxidation and the degradation of PON1 paraoxonase activity were significantly correlated (r = 0.95, p < 0.04).     

Drought-induced spikelet sterility is associated with an inefficient antioxidant defence in rice panicles

Water stress-induced spikelet sterility limits rice production under upland conditions. The causes of spikelet sterility under drought stress are poorly understood. In this study the role of antioxidant defence management in drought-induced spikelet sterility was investigated in two rice ( Oryza sativa ) genotypes differing in drought resistance. Drought-resistant N22 genotype showed less water stress-induced spikelet sterility when compared to the susceptible N118 genotype under upland conditions. The N22 panicles maintained higher RWC and turgor potential and lower H₂O₂ levels across the developmental stages under water stress than that of N118 panicles. Drought-induced enhancement in superoxide dismutase (SOD, EC 1.15.1.1) activity coupled with higher ascorbate (AsA), glutathione (GSH) content and enhanced ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities resulted in lower H₂O₂ levels in N22 panicles. In contrast, insufficient enhancement in SOD, APX and GR activities resulted in relatively higher H₂O₂ levels under water stress in N118 panicles. The N22 panicles exhibited a higher number of SOD and APX isozymes in comparison with N118 panicles that might provide better reactive oxygen species scavenging. Hence it is concluded that well-equipped antioxidant defence plays an important role in minimizing water stress-induced spikelet sterility in upland rice.