Detection of survival motor neuron protein in buccal cells through electrochemiluminescence-based assay.

Spinal muscular atrophy (SMA) is a severe autosomal recessive disorder affecting one in every 10,000 live births. The disease is characterized by loss of alpha-motor neurons in the spinal cord that leads to progressive atrophy and weakness of limb and trunk muscles. This neuromuscular disorder results from deletions and/or mutations within the survival motor neuron 1 (SMN1) gene, leading to a pathologically decreased expression of functional full-length SMN protein. Here we report on the investigation to measure SMN protein levels through electrochemiluminescence immunoassay (ECLIA). This simple assay is a highly quantitative method able to measure SMN protein levels in human, mouse, and rat samples throughout a wide working range with low intra- and interassay error. The sensitivity for human SMN is 30 pg/mL and provides a new tool for the set up of high-throughput screening for basic research. Moreover, we describe a novel tool for a noninvasive assessment of SMN in buccal cells derived from healthy donors, SMA carriers, and SMA patients. The availability of a validated quantitative ECLIA should improve the investigation of novel compounds for the treatment of SMA.

Investigating the role of H2S in 4-HNE scavenging.

4-HNE (4-hydroxy-2-nonenal) is a highly reactive α,ß-unsaturated aldehyde generated from oxidation of polyunsaturated fatty acids and has been suggested to play a role in the pathogenesis of several diseases. 4-HNE can bind to amino acids, proteins, polynucleotides, and lipids and exert cytotoxicity. 4-HNE forms adducts (Michael adducts) with cysteine, lysine, as well as histidine on proteins with the thiol function as the most reactive nucleophilic moiety. Thus, detoxification strategies by 4-HNE scavenging compounds might be of interest. Recently, hydrogen sulfide (H2S) has been identified as an endogenous vascular gasotransmitter and neuromodulator. Assuming that the low-molecular thiol H2S may react with 4-HNE, methods to monitor the ability of H2S to counteract the protein-modifying and cytotoxic activity of 4-HNE are described in this chapter.

Hydrogen sulphide induces HIF-1α and Nrf2 in THP-1 macrophages.

The transcription factor HIF-1α regulates the adaptive response of cells to hypoxia and oxidative stress. In addition, an important regulatory role for HIF-1α in immune reactions and inflammation is suggested. The present study attempts to investigate the effect of the gaseous signalling molecule hydrogen sulphide (H2S) on HIF-1α in THP-1 macrophages using the slow H2S releasing donor GYY4137. We found that H2S induced HIF-1α protein accumulation in THP-1 macrophages in a concentration-dependent manner. Western blot analysis of cell fractions showed that HIF-1α protein translocates into the nucleus and leads to an increase of its target protein glucose transporter-1 (GLUT-1). Activation of nuclear factor-κB (NF-κB), as well as secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced in the presence of H2S. These findings indicate that HIF-1α accumulation due to H2S was not triggered by the NF-κB pathway. The antioxidant pathway Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1) was activated by H2S. Inhibition of the p38 mitogen-activated protein kinase (MAPK) reversed H2S mediated effects, suggesting that the p38 MAPK pathway may be involved in H2S induced HIF-1α/Nrf2 signalling pathways.

The uremic toxin indoxyl sulfate acts as a pro- or antioxidant on LDL oxidation.

Uremic toxins have been shown to play a role in chronic kidney disease (CKD) associated oxidative stress. Oxidative stress and inflammation have been associated with increased risk of cardiovascular disease in uraemia. The oxidative modification of LDL may play a role in early atherogenesis. Enhanced LDL oxidation has been found in uremic patients which may account for accelerated atherosclerosis observed in CKD. The uremic toxin indoxyl sulfate (IS) has been reported to exert oxidative and antioxidative activity. Thus, in the present study we have investigated the influence of IS on the atherogenic modifications of LDL exposed in vitro to different oxidising systems. The transition metal ion (Cu(2) ) and hemin/H2O2 induced lipid oxidation reactions monitored by conjugated diene formation, were inhibited by the presence of IS, which points to possible antioxidant effects by this uremic toxin. A protective effect of IS on LDL apoprotein modification by the exposure to the product of the myeloperoxidase/H2O2/Cl(-) system HOCl, was also observed as estimated by protein carbonyl formation. In contrast, a marked increase in conjugated dienes and lipid hydroperoxides was observed when lipid oxidation was initiated by the free radical generator AAPH in presence of IS. The GC-MS analysis revealed the formation of indole-2,3-dione and 6,12-dihydro-6,12-dioxo-indolo[2,1-b]quinazoline (tryptanthrine) in IS/AAPH reaction. A scheme for the generation of tryptanthrine from IS via indoxyl radicals is proposed, which may facilitate LDL lipid oxidation. Our observations add further insight in the Janus-faced properties of this important uremic toxin.

Known players, new interplay in atherogenesis: Chronic shear stress and carbamylated-LDL induce and modulate expression of atherogenic LR11 in human coronary artery endothelium.

In this study we examined whether low-density lipoprotein (LDL) receptor family members represent a link between blood flow characteristics and modified low-density lipoproteins involved in endothelial injury, a pivotal factor in atherogenesis. We demonstrated the expression of pro-atherogenic LDL receptor relative (LR11) for the first time in human coronary artery endothelial cells (HCAEC) in vitro and in vivo. Next, LR11 expression and regulation were explored in HCAEC cultured conventionally or on the inner surface of hollow fiber capillaries under exposure to shear stress for 10 days in the presence or absence of LDL. There was no LR11 expression under static conditions. When exposed to chronic low shear stress (2.5 dynes/cm²) transmembrane and soluble endothelial-LR11 were detected in high levels irrespective of the type of LDL added (carbamylated or native). In contrast, chronic high shear stress (25 dynes/cm²) inhibited the LR11-inducing effect of LDL such that transmembrane and soluble LR11 expression became non-detectable with native LDL. Carbamylated LDL significantly counteracted this atheroprotective effect of high shear stress as shown by lower, yet sustained expression of soluble and transmembrane LR11. Oxidised LDL showed similar effects compared to carbamylated LDL but caused significantly lower LR11 expression under chronic high shear stress. Medium from HCAEC under LR11-inducing conditions enhanced vascular smooth muscle cell migration, which was abrogated by the anti-LR11 antibody. Expression of LR11 depended entirely on p38MAPK phosphorylation. We conclude that coronary endothelial LR11 expression modulated by LDL and chronic shear stress contributes to atherogenesis. LR11 and p38MAPK are potential targets for prevention of atherosclerosis.

Carbamoylation abrogates the antioxidant potential of hydrogen sulfide.

Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine.

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH(2)-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H(2)O(2)/Cl(-) system is known to react with the NH(2)-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH(2)Cl) was identified as a breakdown product of this reaction.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia.

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.

Hydrogen sulfide scavenges the cytotoxic lipid oxidation product 4-HNE.

Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.

Hydrogen sulfide destroys lipid hydroperoxides in oxidized LDL.

LOOHs (lipid hydroperoxides) in oxLDL [oxidized LDL (low-density lipoprotein)] are potentially atherogenic compounds. Recently, H2S was identified as the third endogenous gasotransmitter in the vasculature. H2O2 is known to be destroyed by H2S. Assuming that H2S may also react with LOOHs, the results show that H2S can destroy LOOHs in oxLDL. The ability of LOOH-enriched LDL to induce HO-1 (haem oxygenase 1) in endothelial cells was abolished by H2S pretreatment. HPLC analysis showed that 9-HPODE [(9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid], a compound found in oxLDL, was reduced to 9-HODE [(9S)-hydroxy-(10E,12Z)-octadecadienoic acid] in the presence of H2S. Thus H2S may act as an antiatherogenic agent by reducing LOOHs to the less reactive LOHs and could abrogate the pathobiological activity of oxLDL.

Hydrogen sulphide: a novel physiological inhibitor of LDL atherogenic modification by HOCl.

Hypochlorite (HOCl), the product of the activated myeloperoxidase/H(2)O(2)/chloride (MPO/H(2)O(2)/Cl(- )) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H(2)S) - which has recently been identified as an endogenous vasorelaxant - to counteract the action of HOCl on LDL. The results show that H(2)S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H(2)S was found to inhibit MPO (one may speculate that this occurs via H(2)S/heme interaction) and destroy H(2)O(2). Thus, H(2)S may interfere with the reactants and reaction products of the activated MPO/H(2)O(2)/Cl(- ) system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: a beneficial "side effect" of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic-phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 micromol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.

The dietary soy flavonoid genistein abrogates tissue factor induction in endothelial cells induced by the atherogenic oxidized phospholipid oxPAPC.

INTRODUCTION: Tissue factor (TF) plays a pivotal role in the generation of thrombin in atherothrombotic disease. The oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC), an active compound of minimally oxidized low-density lipoprotein (MM-LDL), induces TF in endothelial cells (EC). The dietary soybean isoflavonoid genistein has been claimed to reverse several processes leading to atherosclerosis and related cardiovascular events via binding to estrogen receptors, generating nitric oxide (NO) or inhibiting tyrosine kinase-dependent pathways. METHODS AND MATERIALS: The effects and mechanisms of genistein on activity, antigen expression and mRNA levels of oxPAPC-induced TF were studied in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). RESULTS AND CONCLUSIONS: Genistein abrogated oxPAPC-induced TF activity in arterial and venous human EC to basal levels, as measured by functional clotting assay, and downregulated oxPAPC-induced antigen expression measured by flow cytometry and mRNA levels quantified by real-time PCR. Western blotting and inhibitor experiments with the estrogen-receptor inhibitor ICI 182,780 and the NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) showed that the effect may be mediated via inhibition of phosphorylation of ERK, but not upstream MEK1/2. The effect is not mediated by the tyrosine kinase inhibitor activity of genistein, as another tyrosine kinase inhibitor (tyrphostin 25) had no effect. Binding to the estrogen receptor or generation of NO are not involved in the action of genistein on TF. In conclusion genistein reduces oxPAPC-induced TF expression and thereby the prothrombotic phenotype of EC, further substantiating and explaining the beneficial effects of dietary genistein in preventing atherosclerosis and related cardiovascular events.

Cu2+ and Cu+ bathocuproine disulfonate complexes promote the oxidation of the ROS-detecting compound dichlorofluorescin (DCFH).

The water-soluble Cu+ chelator bathocuproine disulfonate (BCS) is widely used to quantify Cu+ or detect Cu+ formation in Cu2+-initiated oxidation reactions. The dichlorofluorescin (DCFH) assay is commonly used to monitor free radical reactions, reactive oxygen species (ROS), or reactive nitrogen species (RNS). Upon oxidation the non-fluorescent DCFH is converted into the fluorescent compound dichlorofluorescein (DCF). In the present communication we show that the Cu+ reagent BCS strongly facilitated the oxidation of DCFH in the presence of Cu2+ or Cu+. In contrast, 2,2'-dipyridyl (DP), which is also a Cu+-complexing reagent, but not as well known and therefore not as commonly used as BCS, did not cause any oxidative modification of DCFH in the presence of Cu2+ or Cu+. We therefore recommend that DP should be used instead of BCS to complex Cu+ in reactions which are initiated by Cu2+ and when ROS/RNS are analyzed by the DCFH oxidation assay.

Products of the reaction of HOCl with tryptophan protect LDL from atherogenic modification.

Hypochlorite (HOCl) attacks amino acid residues in LDL making the particle atherogenic. Tryptophan is prone to free radical reactions and modification by HOCl. We hypothesized, that free tryptophan may quench the HOCl attack therefore protecting LDL. Free tryptophan inhibits LDL apoprotein modification and lipid oxidation. Tryptophan-HOCl metabolites associate with LDL reducing its oxidizability initiated by endothelial cells, Cu(2+) and peroxyl radicals. One tryptophan-HOCl metabolite was identified as 4-methyl-carbostyril which showed antioxidative activity when present during Cu(2+) mediated lipid oxidation, but did not associate with LDL. Indole-3-acetaldehyde, a decomposition product of tryptophan chloramine (the product of the tryptophan-HOCl reaction) was found to associate with LDL increasing its resistance to oxidation. Myeloperoxidase treatment of LDL in the presence of chloride, H(2)O(2) and tryptophan protected the lipoprotein from subsequent cell-mediated oxidation. We conclude that, in vivo, the activated myeloperoxidase system can generate antioxidative metabolites from tryptophan by the reaction of hypochlorite with this essential amino acid.

Sulfite facilitates LDL lipid oxidation by transition metal ions: a pro-oxidant in wine?

Lipid oxidation in LDL may play a role in atherogenesis. It has been shown that sulfite - a compound in the aqueous fraction of wine - could inhibit free radical (AAPH) mediated oxidation of plasma. Thus, sulfite has been proposed as an antioxidant. In contrast, the aqueous phase of wine has recently been shown to contain not fully identified compounds promoting transition metal ion (Cu(2+)) initiated LDL oxidation. As transition metal ions can catalyse the auto-oxidation of sulfite, we studied the influence of sulfite on Cu(2+) initiated LDL oxidation. The results show that sulfite at concentrations found in vivo strongly facilitated LDL oxidation by Cu(2+). The LDL-oxidase activity of ceruloplasmin was also stimulated by sulfite. ROS formation by Cu(2+)/SO(3)(2-) was not inhibited by SOD but by catalase. We propose that formation of Cu(+), sulfite radicals (SO(3)*(-)) and hydroxyl radicals (OH(*)) is a mechanism by which sulfite could act as a pro-atherogenic agent in presence of transition metal ions.

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin, transition metal ions and tyrosyl radicals.

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu(2+)-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe(3+)-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H(2)O(2) was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu(2+)), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H(2)O(2) and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 mumol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification.

OBJECTIVE: Al(3+) stimulates Fe(2+) induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al(3+) and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al(3+) on LDL oxidation. MATERIALS AND METHODS: Using different LDL modifying systems (Fe(2+), Cu(2+), free radical generating compounds, human endothelial cells, hemin/H(2)O(2) and HOCl), the influence of Al(3+) on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation. RESULTS: Al(3+) could stimulate the oxidizability of LDL by Fe(2+), but not in the other systems tested. Al(3+) and Fe(2+) were found to bind to LDL and Al(3+)could compete with Fe(2+) binding to the lipoprotein. Fluorescence polarization data indicated that Al(3+) does not affect the phospholipid compartment of LDL. CONCLUSIONS: The results indicate that increased LDL oxidation by Fe(2+) in presence of Al(3+) might be due to blockage of Fe(2+) binding sites on LDL making more free Fe(2+) available for lipid oxidation.

Free and peptide-bound DOPA can inhibit initiation of low density lipoprotein oxidation.

Hydroxyl radicals have been shown to convert free tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) which has reducing properties. During protein or peptide oxidation such reducing species are also formed from tyrosine residues. Free DOPA or peptide-bound DOPA (PB-DOPA) is able to promote radical-generating events, facilitating the damage of biomolecules such as nucleic acids. Radical induced lipid oxidation in low density lipoprotein (LDL) transforms the lipoprotein into an atherogenic particle. As PB-DOPA has been found in atherosclerotic plaques, we tested the ability of free and PB-DOPA to influence LDL oxidation. Free DOPA, in contrast to tyrosine had strong inhibitory action on both, the copper-ion initiated and metal ion independent (AAPH-induced) lipid oxidation. Free DOPA also inhibited LDL oxidation induced by the copper transport protein ceruloplasmin. To test if PB-DOPA was also able to inhibit LDL oxidation, DOPA residues were generated enzymatically in the model peptides insulin and tyr-tyr-tyr, respectively. PB-DOPA formation substantially increased the ability of both molecules to inhibit LDL oxidation by copper or AAPH. We hypothesize that DOPA-peptides and -proteins may have the potential to act as efficacious antioxidants in the atherosclerotic plaque.