Morphine, a potential inhibitor of myeloperoxidase activity.

Morphine is an opioid alkaloid commonly used in clinical practice for its analgesic properties. The phenolic hydroxyl group of that phenanthrene derivative is pivotal for binding to opioid receptors but it may also be responsible for the antioxidant behavior of morphine reported in several in vitro experiments. In this study, we assessed the effect of morphine on myeloperoxidase (MPO), a hemic enzyme from azurophilic granules of polymorphonuclear neutrophils involved in the production of cytotoxic and microbicidal reactive oxidants during inflammatory response. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling (docking) were performed to study the potential anti-catalytic action of morphine on MPO in comparison with the inhibitory effects of reference antioxidant molecules quercetin, gallic acid and ascorbic acid. The reducing action of morphine on the MPO peroxidase cycle has been investigated using electron paramagnetic resonance (EPR) and UV-visible absorption spectroscopy. Morphine acted as a reducing substrate in the peroxidase cycle of MPO and therefore protected the enzyme against the suicide action of its natural substrate, hydrogen peroxide. The SIEFED experiments associated with the docking study, further demonstrated a lack of strong and sustained anti-catalytic activity of morphine. In summary, from the results of this study, it appears that morphine acts as a weak and reversible inhibitor of MPO that may nonetheless contribute to immunomodulatory and antioxidant effects of this opioid analgesic in vivo.

Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury.

To investigate the role of oxidative stress and/or mitochondrial impairment in the occurrence of acute kidney injury (AKI) during sepsis, we developed a sepsis-induced in vitro model using proximal tubular epithelial cells exposed to a bacterial endotoxin (lipopolysaccharide, LPS). This investigation has provided key features on the relationship between oxidative stress and mitochondrial respiratory chain activity defects. LPS treatment resulted in an increase in the expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 4 (NOX-4), suggesting the cytosolic overexpression of nitric oxide and superoxide anion, the primary reactive nitrogen species (RNS) and reactive oxygen species (ROS). This oxidant state seemed to interrupt mitochondrial oxidative phosphorylation by reducing cytochrome c oxidase activity. As a consequence, disruptions in the electron transport and the proton pumping across the mitochondrial inner membrane occurred, leading to a decrease of the mitochondrial membrane potential, a release of apoptotic-inducing factors and a depletion of adenosine triphosphate. Interestingly, after being targeted by RNS and ROS, mitochondria became in turn producer of ROS, thus contributing to increase the mitochondrial dysfunction. The role of oxidants in mitochondrial dysfunction was further confirmed by the use of iNOS inhibitors or antioxidants that preserve cytochrome c oxidase activity and prevent mitochondrial membrane potential dissipation. These results suggest that sepsis-induced AKI should not only be regarded as failure of energy status but also as an integrated response, including transcriptional events, ROS signaling, mitochondrial activity and metabolic orientation such as apoptosis.

Endotoxin-induced basal respiration alterations of renal HK-2 cells: a sign of pathologic metabolism down-regulation.

To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) both by high-resolution respirometry and electron spin resonance spectroscopy. These two complementary methods have shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells.

ESR technique for noninvasive way to quantify cyclodextrins effect on cell membranes.

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe, which can be further correlated with the microviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes.

Quantification of Randomly-methylated-beta-cyclodextrin effect on liposome: an ESR study.

In the present work, the effect of Randomly-methylated-beta-cyclodextrin (Rameb) on the microviscosity of dimyristoyl-l-alpha phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance (ESR) technique. The ability of Rameb to extract membrane cholesterol was demonstrated. For the first time, the percentage of cholesterol extracted by Rameb from cholesterol doped DMPC bilayer was monitored and quantified throughout a wide Rameb concentration range. The effect of cholesterol on the inner part of the membrane was also investigated using 16-doxyl stearic acid spin label (16-DSA). 16-DSA seems to explore two different membrane domains and report their respective microviscosities. ESR experiments also establish that the presence of 30% of cholesterol in DMPC liposomes suppresses the jump in membrane fluidity at lipids phase-transition temperature (23.9 degrees C).

AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system.

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license.

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells.

Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35+/-0.05x10(7) M(-1) s(-1) for deuteroporphyrin (as embedded source) and 0.8+/-0.04x10(7) M(-1) s(-1) for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.

Fluorescence, absorption and electron spin resonance study of bacteriochlorin a incorporation into membrane models.

Analysis of the bacteriochlorin a absorption spectra suggests the existence of a monomer-dimer equilibrium, particularly intense in phosphate buffer and favored by a decrease of the pH. The dye in methanolic solution is predominantly in monomeric form. Fluorescence and electron spin resonance nitroxide spin labeling measurements indicate that incorporation into the lipid phase of dimyristoyl-L-alpha-phosphatidylcholine liposomes induces dye monomerization. Moreover, the molecules are bound in the external surface of the vesicles and a complete incorporation is ensured by a lipid-to-dye ratio greater than 125.

The antibiotic ceftazidime is a singlet oxygen quencher as demonstrated by ultra-weak chemiluminescence and by inhibition of AAP consumption.

We demonstrated that the cephalosporin antibiotic ceftazidime (CAZ) deactivated singlet oxygen (1O2). We then studied the mechanisms of the CAZ effects on the ultra weak chemiluminescence (uwCL) associated with the energy decay of 1O2 generated by the Mallet reaction (H2O2 + HOCl --> HCl + H2O + 1O2), and on the anthracene-9,10-dipropionic acid (AAP) consumption by 1O2 generated by irradiation of Rose Bengal (RB). The uwCL generated by the Mallet reaction was amplified (6.2 times) by CAZ. The use of red and blue filters, which absorb radiation below 610 nm and between 470 and 700 nm respectively, demonstrated that CAZ increased the uwCL by a radiation emission at wavelengths shorter than the 633 and 704 nm wavelength emissions of 1O2. CAZ was excited by scavenging the energy excess of 1O2, which so returned to its fundamental state, while CAZ deactivated with light emission between 430-480 nm. CAZ also inhibited in a dose-dependent manner the consumption of AAP by 1O2 generated by the irradiation of RB. The protection of AAP by 5 x 10(-3) M CAZ was equivalent to that of 10(-3) M histidine and 3 X 10(-6) M sodium azide. This process of 1O2 deactivation will be useful in diseases characterized by an excessive PMN activation with a release of activated oxygen species.

Hemorrhagic arachnoid cyst with third nerve paresis: CT and MR findings.

We report the CT and MR appearance of a nontraumatic hemorrhagic arachnoid cyst presenting with a third nerve paresis in a 37-year-old man. The cyst, located in the left suprasellar area, contained a fluid-blood level with stigmata of subacute hemorrhage on both CT and MR studies.

Triplet yield of merocyanine 540 in water is wavelength dependent.

Monomers and aggregates of Merocyanine 540 (MC540) in water are able to photoisomerize. The shape of the photoisomer absorption spectrum is very similar to that of the ground state. Triplet state of MC540 in water has been produced by energy transfer from triplet anthracene and displays a broad absorption spectrum between 600 and 700 nm. The triplet state may also be produced by direct excitation of MC540 with UV light. However, when the dye is excited by visible light, no triplet state absorbance in the red could be detected so that the triplet yield of MC540 in water seems to be excitation wavelength dependent.

Electron spin resonance evidence of the generation of superoxide anion, hydroxyl radical and singlet oxygen during the photohemolysis of human erythrocytes with bacteriochlorin a.

Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm2. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D2O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate O2-. and OH. radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH3CHOH. and CO2-. radical adducts, indicating the presence of free OH.. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferrioxamine, suggesting that the iron-catalyzed decomposition of H2O2 was partly involved in the formation of one part of the observed OH.. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with 1O2 production by BCA followed by reaction of 1O2 with DMPO and decay of the intermediate complex to form DMPO-OH and free OH.. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was cell caused by a mixed type 1/type 2 mechanism.

The importance of liposomes as models and tools in the understanding of photosensitization mechanisms.

The various applications of liposomes in understanding photosensitization are described in this paper, with particular emphasis on the various kinds of information that these models allow to obtain in phototherapy. Liposomes are simple vesicles in which an aqueous phase is enclosed by a phospholipidic membrane. They are suitable models mimicking specific situations occurring in vivo and they allow study of the influence of physicochemical, photobiological and biochemical factors on the uptake of photosensitizers by tissues, their mechanisms of action and the subsequent photoinduced tumor necrosis. Moreover, solubilization of the sensitizer into the bilayer seems to improve its tumoral selectivity and its photodynamic efficiency.

Quenching of merocyanine 540 triplet state by nitroxyl radicals in liposomal systems: a laser flash photolysis study.

Laser flash photolysis experiments were undertaken to investigate the interaction between stearic acid nitroxide spin probes and photoexcited merocyanine 540 (MC540) in dimyristoyl-L-alpha-phosphatidylcholine liposomes (membrane model). The measurements of the paramagnetic signal decay kinetics of four different spin-labelled stearic acids (n-DSA) show that the direct interaction between the dye and the probe is affected by the position of the nitroxyl group along the carbon chain. Laser flash photolysis results reveal a significant decrease in the MC540 triplet lifetime in the presence of n-DSA, the effect depending on the depth at which the nitroxyl moiety is localized in the bilayer. Previous results on the rate of disappearance of the electron spin resonance (ESR) nitroxide signal on continuous photolysis of the same systems do not show the same dependence on the localization of the nitroxyl moiety in the liposome. Although the MC540 triplet state seems to be implicated in the reaction process, the results suggest that ESR and laser flash experiments demonstrate two different kinds of mechanism.

Photosensitized production of singlet oxygen by merocyanine 540 bound to liposomes.

The production of singlet oxygen by merocyanine 540 was studied in dimyristoyl-phosphatidylcholine liposomes using two singlet oxygen probes: 9,10-anthracenedipropionic acid (water soluble) and 9,10-dimethylanthracene (liposoluble). Upper and lower limits of singlet oxygen quantum yield for bound merocyanine 540 were determined to be 0.055 and 0.015 respectively. The diffusion characteristics of singlet oxygen were examined using the isotropic enhancement effect of D2O and the inhibitory effect of sodium azide. It was shown that 1O2 spent more than 87% of its lifetime in a vesicle environment. When the singlet-reacting substrate and the dye were both located in the bilayer, approximately 40% of the singlet oxygen remained in the liposomes where it was originally generated.

HIV-1 promoter activation following an oxidative stress mediated by singlet oxygen.

Various biological processes, such as photosensitization or inflammatory reactions, can generate singlet oxygen (1O2) as one of the major oxidative species. Because this oxidant can be generated either extracellularly or intracellularly, it can cause severe damage to various biological macromolecules, even to those deeply embedded inside the cells such as DNA. Sublethal biological modifications induced by different DNA-damaging agents can promote various cellular responses initiated by the activation of various cellular genes and certain heterologous viruses. Since 1O2 fulfils essential prerequisites for a genotoxic substance, we have examined the effects of an oxidative stress, mediated by this species, on cells harbouring a heterologous promoter-leader sequence derived from the human immunodeficiency virus type 1 (HIV-1). Our results demonstrate that HIV-1 long terminal repeat (LTR), integrated into the cellular DNA of epithelial cells, can be transactivated following an oxidative stress mediated by 1O2. In addition, using HIV-1 latently infected promonocytes or lymphocytes, it can be shown that virus reactivation can be induced through a sublethal dose of 1O2 generated intracellularly. An extracellular generation of 1O2 can promote a substantial lethal effect without HIV-1 reactivation. These data may be relevant to the understanding of the events converting a latent infection into a productive one and to the appearance of the acquired immune deficiency syndrome.

Singlet oxygen production and photoisomerization: two competitive processes for merocyanine 540 irradiated with visible light.

The quantum yields of singlet oxygen production by merocyanine 540 have been measured during visible light irradiation performed in methanol and ethanol. These appear to be one hundred times smaller than the quantum yield for rose bengal measured under the same conditions. Flash photolysis experiments demonstrate the ability of merocyanine 540 molecules to isomerize under visible light irradiation: the isomerization quantum yields are about 0.65 in both ethanol and methanol. This information combined with the fluorescence quantum yield data account for the low values for singlet oxygen production.

Increase of the photosensitizing efficiency of the Bacteriochlorin a by liposome-incorporation.

To describe the action mechanisms of Bacteriochlorin a (BCA), a second generation photosensitizer, in phosphate buffer (PB) and in dimyristoyl phosphatidylcholine (DMPC) liposomes we carried out oxygen consumption and ESR measurements. In PB, where BCA was in a monomer-dimer equilibrium, our results suggested that the oxygen consumption was related to the BCA monomers concentration in solution. Incorporation of BCA in DMPC liposomes, by promoting the monomerization of BCA, increased 9-fold the oxygen consumption in comparison to the value in PB. The use of specific singlet oxygen quenchers (Azide and 9,10-Anthracenedipropionic acid) in ESR and oxygen consumption experiments allowed us to assert that BCA was mainly a type II sensitizer when it was incorporated in DMPC. Finally, the cell survival of WiDr cells after a PDT treatment was measured for cells incubated with BCA in cell culture medium and cells incubated with BCA in DMPC. Irrespective of the dye concentration, the cell survival was lower when liposomes were used. This effect could be the result of a better BCA monomerization and/or a different BCA uptake in cells.

Endonasal dacryocystorhinostomy: indications, technique and results.

Dacryocystorhinostomy (DCR) allows an obstructed lachrymal drainage system to be drained into the nasal fossa. Since the development of endonasal microsurgical techniques, the endonasal approach presents itself as an alternative choice to the conventional external approach. It is far less traumatic, yet as efficacious as the conventional approach, and avoids the need for a skin incision and the disruption of the medial canthal structures. Twenty-six patients suffering from lachrymal system obstruction underwent 28 endonasal dacryocystorhinostomies under microscopical (n = 25) or endoscopic control (n = 3). Pre-operative assessment included clinical symptoms (sac swelling, purulent secretions, epiphora) and outflow obstruction on X-rays (conventional dacryocystography and/or subtraction macrodacryocystography). Post-operatively, 23 DCR were free of symptoms. Two presented occasional epiphora and three were unsuccessful. The presence of pre-operative purulent secretions was significantly correlated with post-operative success and with lachrymal sac patency, which is also confirmed by X-ray examination (p<0.001). Thus in cases of purulent secretion with epiphora, X-ray examination is redundant and may be avoided. On the contrary, when epiphora is an isolated symptom, X-rays must be performed in order to determine where the obstruction is located and to provide information on lachrymal sac morphology.

[Diffuse retinal epitheliopathy: study of 30 eyes]. / Epithéliopathie rétinienne diffuse: à propos de 30 yeux.

Retrospective study of 17 patients (30 eyes) with diffuse retinal epitheliopathy followed for at least 5 years. The study is based on clinical data, ophthalmoscopic findings and fluorescein angiograms. The patients (16 men and 1 woman) are all caucasian with a mean age of 43 years. The disease is bilateral in 13 patients. We report an association with systemic corticosteroid treatment after kidney transplantation in 10 cases. Nine patients have arterial hypertension. Two patients are diabetic and 2 present high level of psychological stress. After a mean follow-up of 12 years, a loss of vision of at least 3 lines is noted in 17 eyes. The decrease in vision is related to an enlargement of the pigment epithelium atrophy to the fovea (15 eyes) or with permanent retrofoveolar serous detachment (2 eyes).