Photosensitisation by voriconazole-N-oxide results from a sequence of solvent and pH-dependent photochemical and thermal reactions.

The phototoxicity of voriconazole (VN) prescribed in the treatment of severe fungal infections is frequently reported. Its major metabolite, a N-oxide derivative (VNO), was suspected to be the photosensitizer because it shows a maximum absorbance at ~310 nm in aqueous solutions. It was reported that the VNO photoproduct (VNOP) was phototoxic to human keratinocytes. Steady state and laser flash photolyses were performed to shed light on the phototoxic properties of VNO and VNOP. The quantum yield of the VNOP production by UVB-UVA light in buffered or alcoholic solutions is 0.6. VNOP has been identified as (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-7-oxa-1,3-diazabicyclo[4.1.0]hepta-2,4-dien-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol. VNOP undergoes a marked thermal degradation and an efficient UVA photolysis with well differentiated kinetics and end-products. The temperature-dependent VNOP dark degradation produces a single product VNOPD identified as 6-[(2S,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-5-fluoropyrimidin-4-ol with absorbance maximum at 308 nm and ε = 2700 M-1 cm-1. Under UVB-UVA irradiation, VNOPD, the stable end-product, is a remarkable photodynamic photosensitizer towards Trp and His. The Trp photo-oxidation (Φox(Trp) = 0.13) mainly involves type I radical reactions whereas His is oxidized by 1O2 (Φox(His) = 0.012). These results force us to question the validity of the in vitro photosensitization of human keratinocytes by VNO and VNOP previously reported.

Antibodies directed against annexin A2 and obstetric morbidity.

Acquired and inherited thrombophilia have both been reported to be associated with an increased risk of obstetric complications in early or later stages of pregnancy. Annexin A2 (ANXA2) is strongly expressed in vascular and placental tissues and plays a crucial role in fibrinolysis. The aim of the present study was to evaluate the prevalence of antibodies directed against ANXA2 in patients with recurrent miscarriage or obstetric complications. Anti-ANXA2 antibodies (aANXA2) were detected by ELISA in the sera from 46 women with obstetric morbidity, mainly recurrent miscarriage. The cut-off value for positivity was defined as 3 standard deviations above the mean optical density (OD) obtained in the sera from 42 female blood donors. The prevalence of aANXA2 in patients and healthy individuals was 15.2% and 2.3%, respectively. A statistically significant difference was observed between the 2 groups in terms of aANXA2 IgG titers (p=0.01). The highest aANXA2 levels were observed in sera from 2 patients with recurrent miscarriage and one patient with preeclampsia. aANXA2 could play a role in thrombotic mechanisms leading to recurrent pregnancy loss and placental vascular disease. Further studies are needed to determine whether ANXA2 is critical for maintenance of placental integrity.

Vascular expression of annexin A2 in lupus nephritis.

AIMS: To evaluate vascular expression of annexin A2 (ANXA2) and its subunit S100A10 in lupus nephritis (LN). METHODS: The present histological study included 14 patients with LN and 11 controls (patients with non-lupus kidney diseases). Kidney biopsies from patients with lupus were scored for lupus glomerulonephritis (according to the International Society of Nephrology/Renal Pathology Society 2003 classification) and vascular lesions (such as microthrombi and antiphospholipid syndrome nephropathy (APSN)). ANXA2 and S100A10 expression in glomerular and peritubular capillaries was evaluated by immunohistochemistry on tissue sections. The staining intensity score ranged from 0 (no expression) to 4 (intense expression). RESULTS: In patients with LN, the median age (range) at first kidney biopsy was 36 (18-49). Vascular lesions were observed in six patients (including two with APSN). We observed intense expression of ANXA2 in glomerular and peritubular capillaries while expression of S100A10 was weaker. However, one of the patients with APSN showed strong S100A10 expression. Patients with LN and controls differed significantly in terms of S100A10 expression in peritubular capillaries. We also observed a statistical difference between patients who had LN with renal vascular lesions and those without renal vascular lesions in terms of ANXA2 expression in peritubular capillaries. CONCLUSIONS: The presence of vascular lesions in LN appears to be associated with significant differences in the vascular expression of ANXA2. Vascular expression of ANXA2 was somewhat higher in LN. Vascular expression of S100A10 was somewhat lower in LN (except one of the two patients with APSN). Further studies of ANXA2's putative value as a biomarker of active LN or of vascular lesions in LN are required.

A molecular insight into the phototoxic reactions observed with vemurafenib, a first-line drug against metastatic melanoma.

The electronic properties of vemurafenib (VB) provide a rational basis for understanding its strong UVA-induced phototoxicity. Thus, solvation of hydrophobic VB by hydrogen bonding solvents controls its photophysical, photochemical and photosensitizing properties. Addition of phosphate buffered saline (PBS) to methanol (MeOH) induces a bathochromic shift of the VB absorbance spectrum and a fluorescence emission (λmax = 450 nm, quantum yield (Φ) = 0.011). Phosphorescence (λmax = 461 nm) is observed at 77 K in MeOH. 308 nm laser flash spectroscopy demonstrates that the lifetimes (τ) and quantum yields of the VB triplet state ((3)T(*)(1)) in deaerated MeOH (τMeOH = 0.41 µs, λmax ∼ 380 nm), MeOH-PBS and HSA solutions markedly depend on the microenvironment. A long-lived radical (half-life >200 µs) is also formed. The state (3)T(*)(1) is quenched by O2 and electron donors (Cys and 2'-deoxyguanosine) at a rate constant >1 × 10(9) M(-1) s(-1). UVA-irradiation of VB in air-saturated MeOH or MeOH-PBS solutions produces a UVA-absorbing photoproduct (Φ âˆ¼ 5 × 10(-4)). VB photosensitizes Trp destruction by type I (radical formation) and type II (singlet oxygen ((1)O2) formation) photodynamic reactions (Φ = 0.005). Singlet oxygen production is further demonstrated by the VB-photosensitized His oxidation (ΦMeOH = 0.006).

Chemistry of free radicals produced by oxidation of endogenous α-aminoketones. A study of 5-aminolevulinic acid and α-aminoacetone by fast kinetics spectroscopy.

BACKGROUND: Excess 5-aminolevulinic acid (ALA) and α-aminoacetone (AA) are implicated in ketosis, porphyrinpathies and diabetes. Pathologic manifestations involve O2⁻, H2O2, OH, enoyl radicals (ALA and AA) and their oxidation end products. METHODS: To characterize enoyl radicals resulting from reaction of OH radicals with ALA and AA, micromolar OH concentrations were produced by pulse radiolysis of ALA and AA in aqueous solutions. RESULTS: ALA and AA react with OH at k=1.5 × 109 M⁻¹s⁻¹. At pH7.4, the ALA absorbance spectrum has a maximum at 330 nm (ε=750 M⁻¹cm⁻¹). This band appears as a shoulder at pH8.3 where two ALA species are present: (NH3)⁺-CH2-CO-CH2-CH2-COO⁻ and NH2-CH2-CO-CH2-CH2-COO⁻ (pKa=8.3). At pH8.3, ALA reacts with oxygen (k=1.4 × 108 M⁻¹s⁻¹) but not with O2⁻. At pH8.3, AA oxidation produces two AA species characterized by an absorbance spectrum with maxima at 330 and 450 nm. ALA and AA are repaired by antioxidants (quercetin (QH), catechin, trolox, ascorbate) which are semi-oxidized (k>10(8)M⁻¹s⁻¹). QH bound to HSA or to apoferritin and ferritin repairs ALA and AA. In O2-saturated apoferritin solutions, Q, O2⁻, AA and reaction product(s) react with QH. CONCLUSIONS: The optical absorption properties and the time evolution of ALA and AA were established for the first time. These radicals and their reaction products may be neutralized by antioxidants free in solution or bound to proteins. GENERAL SIGNIFICANCE: Adjuvant antioxidant administration may be of interest in pathologies related to excess ALA or AA production.

On the repair of oxidative damage to apoferritin: a model study with the flavonoids quercetin and rutin in aerated and deaerated solutions.

Ferritin (Ft) impairment through (•)O2(-), H2O2, and (•)OH production occurs in the cases of ketoses, diabetes mellitus, acute intermittent porphyria and tyrosinemia. In addition to (•)Trp and TyrO(•) radical production, ferrous iron liberation and Ft synthesis stimulation, site-specific oxidation reactions are induced leading to toxic iron accumulation in organs with high Ft content, for example, liver and brain. To elucidate the potential pathways to Ft recovery, repair of oxidative damage to horse spleen apoferritin (apoFt) and Ft by quercetin (QH) or rutin (RH) was studied in the presence and absence of oxygen. (•)Trp and TyrO(•) radicals were produced in pulse radiolysis through apoFt oxidation by (•)Br2(-) radicals. QH and RH bind to apoFt on eight sites with binding constants of ˜80,000 and ˜32,000 M(-1), respectively. In deaerated solutions, a repair of apoFt radicals is observed involving both bound and free flavonoids. This repair occurs by a fast intra- and a slow inter-molecular electron transfer from bound and free flavonoids, respectively. With QH, the rate constants are 10(4) s(-1) and 3.5 × 10(7) M(-1) s(-1) for the intra- and intermolecular repair reactions, respectively. Oxygen does not interfere with repair of apoFt or Ft by bound QH but inhibits 90% of Ft repair by RH. These results taken together indicate that flavonoid antioxidants may help alleviate Ft impairment in diseases involving an oxidative stress.

Antibodies against the N-terminal domain of annexin A2 in antiphospholipid syndrome.

Annexin A2 (ANXA2, an endothelial cell receptor for plasminogen and tissue plasminogen activator) has been identified as a new autoantigen in antiphospholipid syndrome (APS). The aim of the present study was to evaluate the presence of antibodies against the N-terminal domain of annexin A2 (ANXA2) in primary APS (PAPS). By using a synthetic peptide corresponding to the 31N-terminal amino acids of ANXA2 (ANXA2(N31)) as an antigen, we performed an enzyme-linked immunosorbent assay (ELISA) to measure anti-ANXA2(N31) IgG and IgM antibodies in the serum of PAPS patients (n=19), systemic lupus erythematosus (SLE) patients (n=50) and healthy blood donors (n=106). We did not find any statistically differences between the three groups in terms of IgG and IgM anti-ANXA2(N31) titres. Elevated IgG anti-ANXA2(N31) titres were not observed in the serum of PAPS or SLE patients who had previously tested positive for anti-ANXA2 antibodies. Thus, the ANXA2 N-terminal domain does not appear to be the target antigen for anti-ANXA2 antibodies in APS.

Serum procalcitonin does not differentiate between infection and disease flare in patients with systemic lupus erythematosus.

Systemic erythematosus lupus (SLE) is a common autoimmune disease. Disease flares may mimic infection with fever, inflammatory syndrome and chills, sometimes resulting in a difficult differential diagnosis. Elevated serum procalcitonin (PCT) levels have been reported to be predictive of bacterial infections, but with conflicting results. The value of serum procalcitonin has not been assessed in large series of SLE. We aimed to describe the distribution of PCT levels in SLE patients with and without flares, to assess the factors associated with increased PCT levels, and to determine the positive and negative predictive values of increased PCT for bacterial infection in SLE patients. Hospitalized SLE patients were included in a retrospective study. Serum PCT had been assayed, or a serum sample had been frozen on admission, before treatment modification. Serum PCT, measured by an automated immunofluorometric assay, and SLEDAI were assessed at the same time. Some 53 women (median age: 33.7 years, range 16-76) and seven men (median age: 52.5 years ± 19) were included. The median SLEDAI for patients with flare (n = 16, 28%) was 2 (range: 0-29). Five patients (8%) had systemic infection. Only one patient had increased PCT levels. Men had significantly higher PCT levels than women (0.196 ± 0.23 versus 0.066 ± 0.03, p < 0.01) and a significant correlation was observed between PCT, age, erythrocyte sedimentation rate, and C-reactive protein. We conclude that PCT levels were within the normal range in infected and non-infected SLE patients and there was no ability to differentiate SLE patients with or without bacterial infection.

Procalcitonin at the onset of giant cell arteritis and polymyalgia rheumatica: the GRACG prospective study.

OBJECTIVES: An epidemic pattern has been reported for GCA and PMR. Immunological studies have shown that an unknown antigen activates the dendritic cells of the adventitia and the type 4 toll-like receptors. Procalcitonin (PCT) is an early marker of bacterial infection. The goal of the study was to assess the level of PCT in GCA and PMR at the onset of the disease. METHODS: Patients diagnosed during the 2002-06 period were randomly selected. All the 46 patients fulfilled the ACR or the Hunder criteria, and all blood samples were taken before steroid therapy. RESULTS: PCT was normal in all patients. PCT was slightly increased in men (0.087 +/- 0.023 microg/l) compared with women (0.066 +/- 0.027 microg/l) (P = 0.009), and in PMR (0.092 +/- 0.027 microg/l) compared with GCA (0.068 +/- 0.026 microg/l) (P = 0.018). There was no significant correlation with inflammation markers. CONCLUSIONS: These results are not in favour of a bacterial trigger for GCA or PMR. Increased PCT levels in patients with inflammatory syndrome, GCA-PMR symptoms and negative temporal artery biopsy may rule out the diagnosis of GCA and PMR.

Anti-annexin II antibodies in systemic autoimmune diseases and antiphospholipid syndrome.

OBJECTIVES: The objective of this study were (1) to evaluate the prevalence of anti-annexin II antibodies in patients with various autoimmune diseases and antiphospholipid syndrome and (2) to correlate anti-annexin II antibodies with anti-phospholipid antibodies. MATERIALS AND METHODS: Anti-annexin II antibodies and anti-phospholipid were detected, using an enzyme-linked immunosorbent assay, in the serum of patients with primary antiphospholipid syndrome (n = 16), systemic lupus erythematosus (n = 53), primary Sjögren syndrome (n = 71), systemic sclerosis (n = 17), systemic vasculitis (n = 18), and rheumatoid arthritis (n = 119). Healthy blood donors (n = 99) were used as controls. RESULTS: Anti-annexin II antibodies were significantly more prevalent in patients with connective tissue diseases (8.5%), especially antiphospholipid syndrome (14.8%) and rheumatoid arthritis (10%), than in controls (2%). An inverse correlation was observed between anti-annexin II antibodies and antiphospholipid antibodies. CONCLUSION: Annexin II can be recognized by antibodies in serum from patients with systemic autoimmune disorders. Further studies are required to determine the clinical significance of anti-annexin II antibodies in rheumatoid arthritis and to determine their diagnostic value in discriminating clinical subgroups of patients with antiphospholipid syndrome.

Impact of inflammation and oxidative stress on vascular calcifications in chronic kidney disease.

Vascular and/or valvular calcifications in patients with chronic kidney disease (CKD) appear to indicate a poor prognosis in terms of overall survival and cardiovascular morbidity and mortality. Inflammation and oxidative stress represent new features of the arterial and/or valvular calcification process. However, only limited observational and epidemiological data are available in these areas. Therefore, the link between inflammation, oxidation and vascular and/or valvular calcifications deserves careful consideration in CKD patients, since they may become targets for the development of new therapeutic strategies.

[Oxidative stress, atherogenesis and cardiovascular risk factors]. / Stress oxydant, athérogenèse et facteurs de risque cardiovasculaire.

Cardiovascular diseases are the main cause of mortality in the western world. It is widely accepted that atherosclerosis, the first etiology, is influenced by free radicals and the oxidizing stress that they cause. In the oxidative theory of atherosclerosis, the atheromatous lesion is initiated by oxidation of two density lipoproteins (LDL), a process still known as lipid peroxidation. Oxidized LDL have many effects on the cells of the vessel wall which, provide an explanation to most of the cellular and tissular changes observed in the plaque. The vascular complications of hypercholesterolaemia, diabetes, hyperhomocysteinemia, hypertension and smoking may, in part, be secondary to oxidizing stress that impairs endothelial function and modify the lipids in the intima of the vessels. The aim of this paper is to review the modes of free radical production, to determine the role of oxidizing stress in the development of atherosclerosis and to show how the different risk factors may initiate atheroma through oxidizing stress.

Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein.

Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs' activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes.

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24 h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitonin-permeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.

Redox reactions of the urate radical/urate couple with the superoxide radical anion, the tryptophan neutral radical and selected flavonoids in neutral aqueous solutions.

The kinetics of several processes involving the potential antioxidant role of urate in physiological systems have been investigated by pulse radiolysis. While the monoanionic urate radical, .UH-, can be produced directly by oxidation with .Br2- or .OH, it can also be generated by oxidation with the neutral tryptophan radical, .Trp, with a rate constant of 2 x 10(7) M-1s-1. This radical, .UH-, reacts with .O2- with a rate constant of 8 x 10(8) M-1s-1. Also, .UH- is reduced by flavonoids, quercetin and rutin in CTAB micelles at rate constants of 6 x 10(6) M-1s-1 and 1 x 10(6) M-1s-1, respectively. These results can be of value by providing reference data useful in further investigation of the antioxidant character of urate in more complex biological systems.

Activation of the JAK/STAT pathway by ceramide in cultured human fibroblasts.

Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1-0.3 U/ml SMase induced a dose-dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose-dependent Tyr-phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose-dependent manner. Concomitantly, SMase enhanced the Tyr-phosphorylation of Janus kinase (JAK) 2, a Tyr-kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr-kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr-phosphorylation, together with STAT1 and STAT3 Tyr-phosphorylation and binding activity. The SMase-induced increase in STAT1/3 binding activity was prevented by methyl-beta-cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER-activated intracellular kinases.

UVA radiation stimulates ceramide production: relationship to oxidative stress and potential role in ERK, JNK, and p38 activation.

Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4-9 J/cm(2). The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.

Oxidized LDL induces an oxidative stress and activates the tumor suppressor p53 in MRC5 human fibroblasts.

It is now well established that oxidized LDL (OxLDL) is involved in the progression of the atheromatous plaque via several mechanisms, including its cytotoxicity toward the arterial wall. Our study demonstrates that a 4-h incubation of cultured human fibroblasts with 25-75 microg/ml OxLDL induced a dose-dependent increase in the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation end products (TBARS). This effect was markedly prevented by the antioxidant vitamin E. The lipid extract of OxLDL partially reproduced the action of the LDL particle itself. Concomitantly, OxLDL enhanced the DNA binding activity of p53 measured by electrophoretic mobility shift assay, and the intracellular protein level of p53 determined by immunoblot analysis. Cycloheximide prevented the OxLDL-induced augmentation in both p53 binding activity and intracellular level. Again, the lipid extract of OxLDL reproduced the effect of OxLDL on p53 binding activity, whereas vitamin E prevented it. These results indicate that OxLDL initiates an intracellular oxidative stress by means of its lipid peroxidation products, leading to the activation of the tumour suppressor p53 by enhancement of p53 protein synthesis. This effect might be related to the cytotoxic effect of OxLDL since the activation of p53 is known to lead to cell cycle arrest, necrosis or apoptosis.

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer.

The kinetics of O2*- reaction with semi-oxidized tryptophan radicals in lysozyme, Trp*(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp*(Lyz) radicals were formed by Br2*- oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 x 10(8) M(-1) s(-1); but drops to 8 x 10(7) M(-1) s(-1) or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp*(Lyz) + Trp*(Lyz) recombination reaction is about (4-7 x 10(6) M(-1) s(-1)) at/or below pH 6.2 then drops to 1.3-1.6 x 10(6) M(-1) s(-1) at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semi-oxidized Trp* residues upon Br2*- reaction. The kinetics of Trp*(Lyz) with ascorbate, a reducing species rather larger than O2*- have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals--leading to the repair of the semi-oxidized Trp*(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp*(Lyz) + O2*- reaction, as is most of the Trp*(Lyz) + Trp*(Lyz) reaction. However, the kinetic behavior of Trp*(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O2*-. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O2*- reactivity. To our knowledge, this is the first report on the kinetics of a protein-O2*- reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.

Alteration of the endocytotic pathway by photosensitization with fluoroquinolones.

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.