Targeting TNFα in severe psoriasis-mass spectrometry reveals a time-dependent specific inhibition of Factor H in responding patients.

Drugs targeting TNFα (eg, Etanercept®) provide effective control of severe psoriasis. In absence of validated biological parameters of inflammation in psoriasis most decisions on therapeutics have relied mostly on clinical criteria, namely the "Psoriasis Area and Severity Index" (PASI). The purpose of this study was to assess by mass spectrometry alterations in concentrations of serum proteins that specifically correlated with effectiveness of Etanercept treatment. This prospective study enrolled 10 patients suffering from moderate to severe psoriasis (PASI score > 10 and < 17) and treated with Etanercept over a period of 24 weeks; 10 healthy, age-matched volunteers provided controls. Serum proteins sensitive to Etanercept treatment were identified using SELDI-TOF (surface-enhanced laser desorption and ionization - time of flight) coupled to nano LC-ESI/MS (nano liquid chromatography-electrospray ionization/tandem mass spectrometry) technologies. For comparisons between groups of individuals p-values (considered significant when < 0.01) were estimated with non-parametric tests, namely Mann-Whitney (for unpaired data) and Wilcoxon signed-rank (for paired data). In responding patients it could be shown using SELDI-TOF spectrometry that two proteins (134 kDa and 4.3 kDa) return to control levels by 24 weeks of treatment. Using nano LC-ESI/MS the 134 kDa species was identified as complement Factor H. These observations deserve further analyses utilizing larger cohorts of patients. Determination of Factor H levels may become a complementary tool to follow remission or predict the onset of relapse in the follow-up of patients under treatment with Etanercept.

Interplay Between Membrane Lipid Peroxidation and Photoproduct Formation in the Ultraviolet A-Induced Phototoxicity of Vemurafenib in Skin Keratinocytes.

According to some authors, the phototoxic response to ultraviolet A (UVA) of patients treated with vemurafenib (VB) may involve VB metabolites. However, the production of singlet oxygen and free radicals and photoproduct formation upon UVA light absorption by the lipophilic VB have been demonstrated. This work is aimed at determining the contribution of reactive oxygen species (ROS), lipid photoperoxidation, and VB photochemistry in the UVA-induced photocytotoxicity in NCTC 2544 keratinocytes. The potent membrane lipid peroxidation effectiveness of VB-photosensitization has been proved by the observation of an effective photohemolysis accompanied by thiobarbituric reactive substances (TBARS) formation in 2% red blood cell (RBC) suspensions. Photohemolysis is inhibited by human serum albumin (HSA) that binds VB and by the antioxidants 2,6-di-tert-butyl-4-methylphenol and Trolox. These data on RBC suggest that VB is readily incorporated in cell membranes and provide clues for understanding the UVA-induced VB-photosensitization of keratinocytes. In keratinocytes, ROS and TBARS formation with 10 µM VB is inhibited by approximately 40% and 50% by 30 µM Trolox and 50 µM vitamin E, respectively, but the light dose-dependent cell survival is unaffected. Whereas cell photokilling depends on the VB concentration, much smaller changes in the lethal doses (LD) than theoretically expected are observed for 25% or 50% cell photokilling when changing absorbed UVA doses and irradiation wavelengths. The lack of antioxidant effect on cell survival and the unexpectedly small LD dependence on absorbed UVA light doses and on irradiation wavelengths strongly suggest that, instead of metabolites, membrane photosensitization and photoproduct formation contribute to the cell photocytotoxicity.

Oxidative stress is independent of inflammation in the neurodegenerative Sanfilippo syndrome type B.

Mucopolysaccharidosis (MPS) type IIIB is a genetic deficiency of α-N-acetylglucosaminidase, inducing accumulation of partially degraded heparan sulfate (HS) oligosaccharides in tissues. In the central nervous system, this accumulation is associated with microglial activation, neurodegeneration, and oxidative stress. We have already shown that HS activates microglial cells through toll-like receptor 4 (TLR4) and triggers neuroinflammation. The present study investigates whether oxidative stress is a direct consequence of inflammation or is an independent event directly caused by HS accumulation. The present study addresses causative links between oxidative stress and inflammation by analyzing the corresponding markers in the cortex of control mice, MPSIIIB mice (with neuroinflammation), and double mutant TLR4 knockout MPSIIIB mice (without neuroinflammation at early stages). Results showed that, although inflammation was not present in the cortex of 10-day-old double mutant MPSIIIB/TLR4(-/-) mice, the enzymatic activity of total superoxide dismutase (SOD) was already greater than in control animals. Moreover, at 3 and 8 months of age, the total enzymatic activities of glutathione peroxidase, SOD, and carbonyl protein levels in the cortex of MPSIIIB/TLR4(-/-) mice were similar to those measured in MPSIIIB mice and were higher than those in controls. The results indicate that the oxidative stress present at a very early stage in the brain of MPSIIIB mice is not the consequence of neuroinflammation. Insofar as it has an impact on the development of neurological disease, reducing oxidative stress might prevent or slow the progression of MPSIIIB.

Increased gut permeability and bacterial translocation after chronic chlorpyrifos exposure in rats.

The epithelium's barrier function is crucial for maintaining homeostasis and preventing the passage of food antigens and luminal bacteria. This function is essentially subserved by tight junctions (TJs), multiprotein complexes located in the most apical part of the lateral membrane. Some gastrointestinal disease states are associated with elevated intestinal permeability to macromolecules. In a study on rats, we determined the influence of chronic, daily ingestion of chlorpyrifos (CPF, a pesticide that crosses the placental barrier) during pre- and postnatal periods on intestinal permeability and TJ characteristics in the pups. Fluorescein isothiocyanate (FITC)-dextran was used as a marker of paracellular transport and mucosal barrier dysfunction. Pups were gavaged with FITC-dextran solution and blood samples were collected every 30 min for 400 min and analyzed spectrofluorimetrically. At sacrifice, different intestinal segments were resected and prepared for analysis of the transcripts (qPCR) and localization (using immunofluorescence) of ZO-1, occludin and claudins (scaffolding proteins that have a role in the constitution of TJs). In rats that had been exposed to CPF in utero and after birth, we observed a progressive increase in FITC-dextran passage across the epithelial barrier from 210 to 325 min at day 21 after birth (weaning) but not at day 60 (adulthood). At both ages, there were significant changes in intestinal TJ gene expression, with downregulation of ZO-1 and occludin and upregulation of claudins 1 and 4. In some intestinal segments, there were changes in the cellular localization of ZO-1 and claudin 4 immunostaining. Lastly, bacterial translocation to the spleen was also observed. The presence of CPF residues in food may disturb epithelial homeostasis in rats. Changes in TJ protein expression and localization may be involved in gut barrier dysfunction in this model. Uncontrolled passage of macromolecules and bacteria across the intestinal epithelium may be a risk factor for digestive inflammatory diseases.

Distribution and quantitation of skin iron in primary haemochromatosis: correlation with total body iron stores in patients undergoing phlebotomy.

Measurement of the concentration of iron in the skin, if correlated with total body iron stores, may enable better informed decisions on when to initiate, change or stop therapy in hereditary heamochromatosis. Naïve haemochromatosis patients with iron overload and with C282Y and/or H63D HFE mutations were evaluated at the following time-points: disease diagnosis, end of the therapy programme, and 6 months after the end of therapy. The distribution and concentration of iron in the skin were assessed by quantitative nuclear microscopy methods, in parallel with serum and plasma iron concentration. Iron content in the liver was determined by nuclear magnetic resonance. Iron accumulated in the epidermis; its concentration increased from outer to inner layers, being maximal in the basal layer (7.33 ± 0.98 µmol/g). At all 3 time-points, most of the iron was associated with the extracellular space. During the phlebotomy programme the iron content of the skin and the liver decreased by a factor of 2. These data suggest that measurements of iron concentration in the epidermis, which is a readily accessible tissue, reflect iron overload in the liver.

Plasma lipoproteins as mediators of the oxidative stress induced by UV light in human skin: a review of biochemical and biophysical studies on mechanisms of apolipoprotein alteration, lipid peroxidation, and associated skin cell responses.

There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces (•)Trp and (•)O2 (-) radicals which trigger lipid peroxidation. Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO(•)) by α -tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α -tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α -tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α -tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed.

The dependence of α-tocopheroxyl radical reduction by hydroxy-2,3-diarylxanthones on structure and micro-environment.

The flavonoid quercetin is known to reduce the α-tocopheroxyl radical (˙TocO) and reconstitute α-tocopherol (TocOH). Structurally related polyphenolic compounds, hydroxy-2,3-diarylxanthones (XH), exhibit antioxidant activity which exceeds that of quercetin in biological systems. In the present study repair of ˙TocO by a series of these XH has been evaluated using pulse radiolysis. It has been shown that, among the studied XH, only 2,3-bis(3,4-dihydroxyphenyl)-9H-xanthen-9-one (XH9) reduces ˙TocO, though repair depends strongly on the micro-environment. In cationic cetyltrimethylammonium bromide (CTAB) micelles, 30% of ˙TocO radicals are repaired at a rate constant of ~7.4 × 10(6) M(-1) s(-1) by XH9 compared to 1.7 × 10(7) M(-1) s(-1) by ascorbate. Water-soluble Trolox (TrOH) radicals (˙TrO) are restored by XH9 in CTAB (rate constant ~3 × 10(4) M(-1) s(-1)) but not in neutral TX100 micelles where only 15% of ˙TocO are repaired (rate constant ~4.5 × 10(5) M(-1) s(-1)). In basic aqueous solutions ˙TrO is readily reduced by deprotonated XH9 species leading to ionized XH9 radical species (radical pK(a) ~10). An equilibrium is observed (K = 130) yielding an estimate of 130 mV for the reduction potential of the [˙X9,H(+)/XH9] couple at pH 11, lower than the 250 mV for the [˙TrO,H(+)/TrOH] couple. A comparable value (100 mV) has been determined by cyclic voltammetry measurements.

Evidence for a slow and oxygen-insensitive intra-molecular long range electron transfer from tyrosine residues to the semi-oxidized tryptophan 214 in human serum albumin: its inhibition by bound copper (II).

A slow, long range electron transfer (SLRET) in human serum albumin (HSA) is observed from an intact tyrosine (Tyr) residue to the neutral tryptophan (Trp) radical (Trp·) generated in pulse radiolysis. This radical is formed, at neutral pH, through oxidation with Br (2) (·-) radical anions of the single Trp 214 present. The SLRET rate constant of ~0.2 s(-1) determined is independent of HSA concentration and radiation dose, consistent with an intra-molecular process. This is the slowest rate constant so far reported for an intra-molecular LRET. In sharp contrast with the LRET reported for other proteins, the SLRET observed here is insensitive to oxygen, suggesting that the oxidized Trp is inaccessible to-or do not react with radiolytically generated O (2) (·-) . In N(2)O-saturated solutions, the SLRET is inhibited by Cu(2+) ions bound to the His 3 residue of the N-terminal group of HSA but it is partially restored in O(2)-saturated solutions.

Structure-activity relationships in hydroxy-2,3-diarylxanthone antioxidants. Fast kinetics spectroscopy as a tool to evaluate the potential for antioxidant activity in biological systems.

A structure-activity relationship has been established for eight hydroxy-2,3-diarylxanthones (XH) bearing hydroxy groups on the two aryl rings. One-electron oxidation by superoxide radical-anions (˙O(2)(-)) and ˙Trp radicals as well as reaction with ˙CCl(3)O(2) and ˙CHCl(2)O(2) radicals demonstrates that two OH groups are required for efficient antioxidant reactivity in cetyltrimethylammonium bromide micelles. Hydroxy groups at the meta and para positions on either of the two phenyl rings confer enhanced reactivity, but XH bearing an OH at the para position of either phenyl ring is unreactive. While oxidation is favoured by OH in both meta and para positions of 2-aryl xanthone substituents, addition of a third and/or fourth OH enhances electron-donating capacity. In Cu(2+)-induced lipid peroxidation of human LDL, the lag period preceding the commencement of lipid peroxidation in the presence of XH bearing OH at meta and para positions on the 3-phenyl ring is extended to twice that observed with a comparable concentration of quercetin, a reference antioxidant. These antioxidants are also superior to quercetin in protecting human skin keratinocytes against tert-butylhydroperoxide-induced oxidative stress. While XH antioxidant activity in model biological systems is consistent with the structure-activity relationship, their response is also modulated by the localization of XH and by structural factors.

Uremic toxin indoxyl sulfate inhibits human vascular smooth muscle cell proliferation.

Uremic syndrome is attributed to the progressive retention of a large number of compounds, such as indoxyl sulfate, which under physiological conditions are excreted by the kidneys. Previous in vitro studies have demonstrated that uremic indoxyl sulfate concentrations induce a weak increase in the proliferation of both rat and human vascular aortic smooth muscle cells (hVASMC) after short term exposition to the toxin (i.e. 24 h). In the present study, we evaluated indoxyl sulfate effects on the proliferation of hVASMC at three different concentrations after long-term exposure (seven days). In contrast to previously published studies, we observed a dose-dependent and significant inhibitory effect of this toxin on hVASMC proliferation. We also demonstrated that indoxyl sulfate inhibits epidermal growth factor-induced hVASMC proliferation after long-term exposure. Indoxyl sulfate effects were associated with a dose-dependent induction of intracellular reactive oxygen species and up-regulation of p21 and p27 protein expression. Chronic exposure to indoxyl sulfate produces a significant inhibitory effect on hVASMC proliferation. The relevance of these findings must be evaluated by further studies, particularly in an in vivo setting.

Chain-dependent photocytotoxicity of tricationic porphyrin conjugates and related mechanisms of cell death in proliferating human skin keratinocytes.

Photodynamic therapy (PDT) is a poor treatment option for nodular basal cell carcinomas and squamous cell carcinomas. As a result, the search for new photosensitizers with better effectiveness is of current interest. The photocytotoxicity of conjugates (P-R) of a water-soluble tri-cationic porphyrin (P-H) having similar efficiency of production of singlet oxygen, the PDT cytotoxin, has been assessed in vitro. Links between uptake, intracellular localization, photooxidative stress, photocytotoxicity and ability to induce programmed cell death are established. Conjugates bearing methyl (P-Me), Di-O-isopropylidene-(-d-galactopyranosyl (P-OGal) or N,N'-dicyclohexylureidooxycarbonyl (P-DDC) chains are efficiently taken-up by proliferating NCTC 2544 keratinocytes. The relative order of photocytotoxicity is P-OGal >P-DDC=P-Me≫P-H. The photocytotoxic potential of P-Me, P-OGal and P-DDC equals that of endogenous protoporphyrin IX induced by δ-aminolevulinic acid or its esters, the pro-drugs currently employed for PDT of skin lesions. Microfluorometry shows that P-Me, P-OGal, and P-DDC localize in endocytotic or pinocytotic vesicles but not in mitochondria or nucleus. Absence of annexin V binding, caspase activation or chromatin condensation suggests that cell photosensitization by P-R does not induce apoptosis. On the other hand, P-OGal photocytotoxicity correlates with appearance of multiple vesicles that have hallmarks of autophagy compartments, being decorated with the marker LC3 in cells transfected with an expression vector encoding GFP-LC3. p38 and JNK phosphorylation and inhibition of ERK1/2 phosphorylation suggest close relationship between mortality of NCTC 2544 keratinocytes and MAPK pathway impairment. Given their potentially easy formulation, water-soluble P-R are promising powerful photosensitizers for PDT of skin lesions.

Tricationic porphyrin conjugates: evidence for chain-structure-dependent relaxation of excited singlet and triplet States.

Conjugates of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridinium-4-yl)porphyrin (P-H) are promising photoactive agents for medical applications. As their ultimate efficacy will depend on the behavior of initial excited states, photophysical parameters have been determined with conventional steady-state absorption and fluorescence as well as time-resolved femto- and nanosecond spectroscopies. The fluorescence quantum yield of P-H and P-H conjugated to uncharged groups increases from approximately 0.03 in pH 7 buffer to approximately 0.05 in Triton X100 micelles (TX100) and in ethanol and to 0.12 in sodium dodecyl sulfate (SDS) micelles. Corresponding (1)S(1) lifetimes are approximately 5-10 ns. In buffer, an equilibrium between P-H monomers and small-size aggregates is observed. Conjugation with poly-S-lysine (P-(Lys)(n)) results in fluorescence quenching in all solvents. Structural reorganization of conjugates bearing a Di-O-isopropylidene-alpha-d-galactopyranosyl or a alpha/beta-d-galactopyranosyl group occurs in ethanol (k approximately 0.15 ps(-1)) after (1)S(1) state solvation (approximately 700 fs). Relaxation of bulky P-(Lys)(n) polypeptide chains takes place on a longer time scale in all solvents (k

The alkyl chain length of 3-alkyl-3',4',5,7-tetrahydroxyflavones modulates effective inhibition of oxidative damage in biological systems: illustration with LDL, red blood cells and human skin keratinocytes.

It is shown that the relationship between the alkyl chain length of 3-alkyl-3',4',5,7 tetrahydroxyflavones (FnH) bearing alkyl chains of n=1, 4, 6, 10 carbons and their capacity to counter oxidative damage varies markedly with the nature of the biological system. In Cu(2+)-induced lipid peroxidation of low-density lipoprotein (LDL), the less hydrophobic short-chain F1H and F4H are probably located in the outer layer of LDL and parallel the reference flavonoid antioxidant, quercetin (Q) as effective inhibitors of lipid peroxidation. A marked inhibition of haemolysis induced in red blood cells (RBC) suspensions by the membrane-permeant oxidant, tert-butylhydroperoxide (t-BuOOH), is observed with F4H and F6H present at concentration in the micromolar range. However, F10H the most hydrophobic FnH is even more effective than Q against both haemolysis and lipid peroxidation as measured by malondialdehyde (MDA) equivalents. In oxidation of RBC by H(2)O(2,) at least 50 times more F6H and F10H than by t-BuOOH are required to only partly inhibit haemolysis and MDA production. The F1H, F4H and Q are found rather inactive under these conditions. At concentrations in the micromolar range, a marked protection against the cytotoxic effects of the t-BuOOH-induced oxidative stress in human skin NCTC 2544 keratinocytes is also exhibited by the four FnH antioxidants and is comparable to that of Q. Thus, the four FnH species under study may be considered as potent antioxidants which manifest complementary anti-oxidative actions in biological systems of markedly different complexity.

Photodynamic therapy: Dermatology and ophthalmology as main fields of current applications in clinic.

Photodynamic therapy (PDT) of skin tumors or pre-cancerous lesions and of age-related macular degeneration combines the administration of porphyrins or porphyrin precursors and illumination with red light at the diseased sites. Photosensitizers absorbing light beyond 630 nm where tissues have the highest transmittance produce singlet oxygen, a highly reactive activated oxygen species and a major cytotoxin. The PDT of age-related macular degeneration is performed with red laser light after i.v. injection of verteporfin (Visudyne) a hydrophobic porphyrin carried by serum lipoproteins whose endocytosis leads to accumulation of the porphyrin in endothelial cells of choroidal neo-vessels. In the PDT of skin cancers, local synthesis of the photosensitizer occurs after topical application of the natural protoporphyrin IX precursor delta-aminolevulinic acid (or its ester forms) on the lesions. In all the cases, the photosensitizers should be rapidly excreted to avoid a long lasting skin photosensitivity.

One-electron reduction of superoxide radical-anions by 3-alkylpolyhydroxyflavones in micelles. Effect of antioxidant alkyl chain length on micellar structure and reactivity.

In micellar solutions, one-electron reduction of *O2(-) radical-anions by 3-alkylpolyhydroxyflavones (FnH) with alkyl chains of n = 1, 4, 6, 10 carbons produces phenoxyl radicals ( (*Fn) identical to those obtained by one-electron oxidation by *Br2(-) radical-anions or by repair of tryptophan radicals. In cetyltrimethylammonium bromide (CTAB), F1H localizes in the Stern layer, and alkyl chains of other FnH solubilize in the hydrophobic interior, interacting with cetyl tails. This interaction produces more compact micelles with lower intramicellar fluidity, as suggested by the increase in the pseudo-first-order rate constant of *Fn formation ( k 1) from approximately 390 s (-1) for n = 1 to 610 s (-1) for n = 10, leading to an intramicellar bimolecular rate constant of 1 x 10 (5) M (-1) s (-1). Additionally, *F1 and *F4 decay by intermicellar bimolecular reaction (2 k = 20 and 2 x 10 (5) M (-1) s (-1), respectively) whereas other *Fn radicals are stable over seconds due to increased localization with regards to the Stern layer. In contrast, the thick uncharged hydrophilic palisade layer and the compact hydrophobic core of Triton X100 micelles are responsible for a much higher microviscosity resulting in a decrease in k 1 from approximately 15.6 s (-1) for n = 1 to 9.6 s (-1) for n = 10.

Photophysical properties of a photocytotoxic fluorinated chlorin conjugated to four beta-cyclodextrins.

A meso-tetrakis(pentafluorophenyl)-chlorin with the reduced pyrrole ring linked to an isoxazolidine ring (FC) has been conjugated to four beta-cyclodextrins (CDFC). The CDFC exhibits excellent water solubility and is a potent photosensitizer towards proliferating NCTC 2544 human keratinocytes. The study by conventional steady state absorption and fluorescence spectroscopies and by time-resolved femto- and nanosecond laser flash spectroscopies suggests that in ethanol and pH 7 buffer the beta-cyclodextrins embed the highly hydrophobic tetrakis(pentafluorophenyl)-chlorin macrocycle and strongly interact with the chlorin rings in the singlet and triplet manifolds. In these solvents, femtosecond spectroscopy suggests that the conjugate undergoes a rapid relaxation in the upper excited singlet states induced by photochemical and/or conformation change(s) at a rate of about 5 ps(-1) to fluorescent states whose lifetime is approximately 8 ns. This interaction is destroyed upon addition of Triton X100 to buffer. Both FC and CDFC strongly fluoresce (Phi(F) approximately 0.5) in micelles. Similar behavior is observed at the triplet level. In ethanol and water, the initial transient triplet state absorbance decays within 1-3 mus yielding a longer lived triplet with spectral properties indistinguishable from that of original difference absorbance spectra. The determination of the molar absorbance in the 440-460 nm region ( approximately 35 000 M(-1) cm(-1)) leads to an estimate of approximately 0.2 for the triplet formation quantum yield of FC in toluene and of FC and CDFC in Triton X100 micelles. Quenching of the CDFC triplets by dioxygen in buffer produces (1)O(2) in a good yield consistent with the effective photocytotoxicity of the chlorin-cyclodextrins conjugate towards cultured NCTC 2544 human keratinocytes. By contrast, FC which aggregates in buffer produces little if any (1)O(2).

High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells.

In this work, we investigated the effect of inorganic phosphate (Pi) on the differentiation of monocyte/macrophage precursors into an "osteoclastic" phenotype, and we delineated the molecular mechanisms which could be involved in this phenomenon. This was achieved by stimulating human peripheral blood monocytic cells and RAW 264.7 monocyte-macrophage precursor cells to differentiate into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). RANKL has been previously reported to stimulate the signaling kinases ERK 1/2, p38, Akt, JNK, and the DNA-binding activity of the transcription factors AP-1 and NF-kappaB. Increase in extracellular Pi concentration (1.5-4.5 mM) dose-dependently inhibits both osteoclastic differentiation and bone resorption activity induced by RANKL and M-CSF. Pi was found to specifically inhibit the RANKL-induced JNK and Akt activation, while RANKL-induced p38 and ERK 1/2 phosphorylation were not significantly affected. Moreover, we found that Pi significantly reduced the RANKL-stimulated DNA-binding activity of NF-kappaB. The effects of Pi on osteoclast differentiation and DNA-binding activity of NF-kappaB were prevented by Foscarnet, a sodium-phosphate cotransport inhibitor, suggesting that the effects of Pi occur subsequently to its intake. These results demonstrate that Pi downregulates the differentiation of osteoclasts via a negative feedback exerted on RANK-RANKL signaling.

Albumin-bound quercetin repairs vitamin E oxidized by apolipoprotein radicals in native HDL3 and LDL.

In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopheroxyl radical (alphaTocO*). Fast spectroscopic studies show that two populations representing 80% of alphaTocO* initially formed are repaired over several seconds with rate constants of 3.0 x 10(6) and 1.5 x 10(5) M-1 s-1 by quercetin bound to human serum albumin (HSA) at physiologically relevant concentration. Formation of HSA-bound quercetin radicals (*Qb) is observed. In the major fraction of HDL3 particles lacking alphaTocOH, TyrO* and *Trp are repaired by free and HSA-bound quercetin. In LDL particles which all contain alphaTocOH, alphaTocO* radicals are formed in the millisecond time scale by repair of TyrO* radicals produced in apolipoprotein B. Then, 75% of initial alphaTocO* are repaired over seconds by HSA-bound quercetin (rate constant: 2.0 x 10(6) M-1 s-1). HSA-bound quercetin can also repair *Trp radicals. In O2-saturated solutions, the fraction of alphaTocO* radicals (more than 50%) not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of *Qb but to a much lesser extent in LDL than in HDL.

Interplay of oxygen, vitamin E, and carotenoids in radical reactions following oxidation of Trp and Tyr residues in native HDL3 apolipoproteins. Comparison with LDL. A time-resolved spectroscopic analysis.

It has been recently shown that the inhibition of apolipoprotein A-I (apoAI) reverse cholesterol transport activity during oxidation of HDL by myeloperoxidase may involve myeloperoxidase electron transfer pathways other than those leading to tyrosine chlorination. To better understand how such mechanisms might be initiated, the role of semioxidized Tyr and Trp residues in loss of apoAI and apolipoprotein A-II (apoAII) integrity has been assessed using selective Trp and Tyr one-electron oxidation by *Br2(-) radical-anions in HDL3 as well as in unbound apoAI and apoAII. Behavior of these radicals in apolipoprotein B of LDL has also been assessed. Formation of semioxidized Tyr in HDL3 is followed by partial repair during several milliseconds via reaction with endogenous alpha-tocopherol to form the alpha-tocopheroxyl radical. Subsequently, 2% of alpha-tocopheroxyl radical is repaired by HDL3 carotenoids. With LDL, a faster repair of semioxidized Tyr by alpha-tocopherol is observed, but carotenoid repair of alpha-tocopheroxyl radical is not. Only a small fraction of HDL3 particles contains alpha-tocopherol and carotenoids, which explains limited repair of semioxidized Tyr by alpha-tocopherol. All LDL particles normally contain multiple alpha-tocopherol and carotenoid molecules, and the lack of repair of alpha-tocopheroxyl radical by carotenoids probably results from hindered mobility of carotenoids in the lipid core. Western blots of gamma-irradiated HDL3 comparable to those reported for apoAI myeloperoxidase oxidation show that the incomplete repair of semioxidized Tyr and Trp induces apoAI and apoAII permanent damage including formation of a heterodimer of one apoAI with a monomeric apoAII at about 36 kDa.