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See detailJNK/ROS Signaling Pathway Is Responsible for Induction of Autophagy in HDAC5 depleted Cancer Cells
Hendrick, Elodie ULg; Mathéus, Nicolas; Peixoto, Paul ULg et al

Conference (2013, January 29)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis (ref papier). The study of autophagy in cancer is a new research field that has recently generated tremendous attention due to the recognition that autophagy can have either pro-survival or pro-death functions depending on its level of activation. In addition, more and more studies indicate that a complex relationship exists between autophagy and apoptosis, and that the interplay between these two processes determines whether a cell will live or die. Aims: The goal of this study is to further understand the role of autophagy induced by HDAC5 depletion. Current investigations include determining the molecular mechanisms by which HDAC5 depletion induces autophagy and exploring regulatory relationship between autophagy and apoptosis on cancer cell death in absence of HDAC5. Results: The set up of the autophagy in absence of HDAC5 was demonstrated by the conversion of LC3 and development of autophagosomes by electronic microscopy. Transcriptomic study demonstrated a deregulation of a set of genes involved in ROS detoxification in HDAC5 depleted cancer cells leading to significant increase of ROS levels. Further investigations showed that pretreatment with NAC, a ROS scavenger, effectively blocked the accumulation of ROS and autopahgy triggered by HDAC5 silencing. Moreover, HDAC5 depletion induces activation of JNK, and knockdown of JNK by siRNA inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by HDAC5 depletion indicating that JNK activation may be a upstream signaling of ROS and should be a core component in HDAC5 silencing-induced autophagic signaling pathway. Finally, blocking of autophagy induced by HDAC5 silencing with NAC or chloroquine and bafilomycin enhanced pro-apoptotic effect. Conclusion: Autophagy functions as a prosurvival mechanism to mitigate HDAC5 depletion-induced apoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of specific HDAC5 inhibition. [less ▲]

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See detailJNK/ROS signaling pathway is responsible for induction of autophagy in HDAC5 depleted cancer cells
Hendrick, Elodie ULg; Mathéus, Nicolas; Peixoto, Paul ULg et al

Poster (2013, January 28)

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure ... [more ▼]

Introduction: Histone deacetylases (HDAC) is a family of eighteen enzymes which modulates the acetylation level of histones and non-histone proteins to regulate gene expression and chromatin structure. Broad spectrum inhibitors of these enzymes such as SAHA can inhibit tumor growth both in vitro and in vivo and are currently used as anti-cancer agents in clinic. For many years, we are investigating the specific role of individual HDAC members in cancer biology and we have recently demonstrated that depletion of HDAC5 using siRNA technology triggered cancer cells to both autophagy and apoptosis (ref papier). The study of autophagy in cancer is a new research field that has recently generated tremendous attention due to the recognition that autophagy can have either pro-survival or pro-death functions depending on its level of activation. In addition, more and more studies indicate that a complex relationship exists between autophagy and apoptosis, and that the interplay between these two processes determines whether a cell will live or die. Aims: The goal of this study is to further understand the role of autophagy induced by HDAC5 depletion. Current investigations include determining the molecular mechanisms by which HDAC5 depletion induces autophagy and exploring regulatory relationship between autophagy and apoptosis on cancer cell death in absence of HDAC5. Results: The set up of the autophagy in absence of HDAC5 was demonstrated by the conversion of LC3 and development of autophagosomes by electronic microscopy. Transcriptomic study demonstrated a deregulation of a set of genes involved in ROS detoxification in HDAC5 depleted cancer cells leading to significant increase of ROS levels. Further investigations showed that pretreatment with NAC, a ROS scavenger, effectively blocked the accumulation of ROS and autopahgy triggered by HDAC5 silencing. Moreover, HDAC5 depletion induces activation of JNK, and knockdown of JNK by siRNA inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by HDAC5 depletion indicating that JNK activation may be a upstream signaling of ROS and should be a core component in HDAC5 silencing-induced autophagic signaling pathway. Finally, blocking of autophagy induced by HDAC5 silencing with NAC or chloroquine and bafilomycin enhanced pro-apoptotic effect. Conclusion: Autophagy functions as a prosurvival mechanism to mitigate HDAC5 depletion-induced apoptotic cell death, suggesting that targeting autophagy might improve the therapeutic effects of specific HDAC5 inhibition. [less ▲]

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See detailIntra- and extracellular antioxidant capacities of the new water-soluble form of curcumin (NDS27) on stimulated neutrophils and HL-60 cells
Derochette, Sandrine ULg; Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg et al

in Chemico-Biological Interactions (2013), 201(1-3), 49-57

Phagocytic cells, especially neutrophils (PMNs) are specialized in the production of reactive oxygen species (ROS) to kill pathogenic agents, but an excessive ROS production is associated with tissue ... [more ▼]

Phagocytic cells, especially neutrophils (PMNs) are specialized in the production of reactive oxygen species (ROS) to kill pathogenic agents, but an excessive ROS production is associated with tissue damages and inflammatory diseases. Phagocytes are thus prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenols to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27, a highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs and human promyelocytic leukemia cells (HL-60). NDS27 was either pre-incubated with cells and eliminated before their activation (intracellular effect) or let in the medium (extracellular effect). Our results indicate that NDS27 significantly and dose-dependently (10 6 M–10 4 M) inhibited the ROS production in both cell types without affecting their viability. NDS27 was able to cross and interact with cell membrane, especially for HL-60 cells, while we observed a better intracellular antioxidant effect with PMNs. The activity of myeloperoxidase (MPO) released by PMNs and HL-60 cells, was decreased by NDS27, but more efficiently for PMNs. These results suggested that the greater efficiency of NDS27 in PMNs is due to an inhibitory effect on cells which are more mature for ROS production, probably by targeting the enzymes implied in respiratory burst like MPO. The modulatory effect of NDS27 on the oxidant activity of cells involved in immune and inflammatory responses opens perspectives for a therapeutic control of pathologies with excessive inflammatory reactions. [less ▲]

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See detailEquine myeloperoxidase: A novel biomarker in synovial fluid for the diagnosis of infection.
Wauters, J.; Pille, F.; Martens, A. et al

in Equine Veterinary Journal (2013), 45(3),

REASONS FOR PERFORMING STUDY: Equine joint infection is a life-threatening disorder, and confirmation of the diagnosis can be difficult. Synovial fluid biomarkers may assist the discrimination between ... [more ▼]

REASONS FOR PERFORMING STUDY: Equine joint infection is a life-threatening disorder, and confirmation of the diagnosis can be difficult. Synovial fluid biomarkers may assist the discrimination between infectious and noninfectious joint disease. OBJECTIVES: This study investigates whether the immunological detection of total and enzymatically active myeloperoxidase (MPO) assists the diagnosis of joint infection in horses. METHODS: The following 4 sample groups were included: healthy; osteochondritis dissecans (OCD); traumatic synovitis; and culture-confirmed infected joints. Synovial fluid was analysed for total MPO by a horse-specific sandwich enzyme-linked immunosorbent assay (ELISA) and for active MPO using the specific immunological extraction followed by enzymatic detection (SIEFED) technique. Western blot analysis was performed to confirm the antibody specificity. RESULTS: Synovial fluid from infected joints contained significantly more total and active MPO than samples from healthy joints, joints affected by OCD and joints with traumatic synovitis. Cut-off values were set at 5000 and 350 ng/ml for total and active MPO, respectively, with fair sensitivity, specificity, positive and negative predictive values and likelihood ratios for infection. Correlation coefficients were reported between the total as well as the active MPO levels and the routine synovial fluid parameters, i.e. the white blood cell count, the neutrophil count and the total protein level. No correlation was observed between MPO and either the age of the horse or the joint affected. Western blotting confirmed the antibody specificity for equine MPO. CONCLUSIONS AND POTENTIAL RELEVANCE: Synovial fluid MPO was identified as a very promising biomarker to augment the discrimination of infectious vs. noninfectious joint disease in horses. Both ELISA and SIEFED techniques can be used for its specific and rapid detection. The analysis of synovial fluid MPO can be used as a complementary test to aid in the discrimination between infectious and noninfectious joint disease, especially when the white blood cell counts and the total protein level are inconclusive. [less ▲]

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See detailSystemic Active and Total Myeloperoxidase Levels in Coronary Artery Disease.
GACH, Olivier ULg; MAGNE, Julien ULg; Franck, Thierry ULg et al

in Cardiology (2013), 126(Suppl. 2), 1-521

Backgound: Measurement of total Myeloperoxidase (MPO) by ELISA is considered as a marker of neutrophil activation but is not the true indicator of the degree of its activity. In a dynamic pathology such ... [more ▼]

Backgound: Measurement of total Myeloperoxidase (MPO) by ELISA is considered as a marker of neutrophil activation but is not the true indicator of the degree of its activity. In a dynamic pathology such as atherosclerosis, it may be important to measure the real active part of MPO because it represents the true witness of the oxidant potential of the enzyme. Aim: To identify the relation between coronary artery disease identified by coronaro-angiography on measured serum total and active MPO levels and evaluate the correlation between these MPO levels and the presence of clinically defined unstable condition. Methods: Prospective analyse of serum samples of patients before (within 30 min) coronaro-angiography. Total and active MPO concentrations were assessed by sandwich Elisa and SIEFED® method’s respectively. Results: Two hundred and twenty patients were included in this study (age: 66.1±10.7 years, 67% of male). Among these, 62% presented significant coronary artery disease (stenosis more than 60% at least in one épicardial coronary artery). Twenty four patients (11%) presented unstable coronary syndrome. Mean active and total MPO in the general population were 50.1±63.5 and 147.6±223.3 ng.mL-1 respectively. In comparison, mean active MPO was 47.1±47.9 ng.mL-1 in stable patients and 75.1±135.2 ng.mL-1 in unstable patients (p=0.04). Mean total MPO was 146.3±224.7 ng.mL-1 in stable patients and 158.2±215.8 ng.mL-1 in the unstable’s one (p=0.8). There was a significant correlation between active MPO levels and instability (r=0.14, p=0.04) not present for total MPO levels (r=0.016, p=0.8). Conclusion: We observed a correlation between active MPO and clinical instability while there was no correlation with total MPO. Our preliminary results suggest that this marker could be a powerful indicator of instability which could possess an important prognostic impact. This hypothesis requires an evaluation in wider population and during a prolonged follow-up. [less ▲]

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See detailDo developmental orthopaedic disorders influence future jumping performances in Warmblood stallions?
Verwilghen, D. R.; Janssens, S.; Busoni, Valeria ULg et al

in Equine veterinary journal (2013), 45(5), 578-81

REASONS FOR PERFORMING THE STUDY: Few reports are available on the relationship between developmental orthopaedic diseases (DOD) and future performances in Warmblood horses. OBJECTIVES: To investigate the ... [more ▼]

REASONS FOR PERFORMING THE STUDY: Few reports are available on the relationship between developmental orthopaedic diseases (DOD) and future performances in Warmblood horses. OBJECTIVES: To investigate the relationship between performance and the presence of DOD lesions. METHODS: Records of Warmblood stallions for which radiographic and performance data were available were collected. Showjumping performances were expressed as scores derived from the final ranking of horses in each competition. These scores are available in an established performance database. The relationship between radiographic findings and both performance scores and number of performances was analysed using a linear regression model. RESULTS: Two hundred and fifteen horses met the inclusion criteria. There was no difference in either the number of performances or performance score between horses categorised as affected with DOD lesions (independent of joint location) compared with controls. Significantly lower numbers of performances were recorded for horses with osteochondral fragments (OCD) located at the dorsal aspect of the sagittal ridge of the metacarpo/metatarsophalangeal bone. No significant difference was found between horses affected with DOD lesions of the tarsocrural joint and controls. Horses with osteochondrosis of the lateral trochlear ridge of the femur had both significantly lower performance scores and numbers of performances compared with controls. CONCLUSION: This study demonstrated that specific DOD location and site within the joint have an influence on performance. Osteochondral fragments in the femoropatellar and at the dorsal aspect of the sagittal ridge of the metacarpo/metatarsophalangeal joint resulted in lowered performance. Fragmentation in the tarsocrural joint had no influence on performance. POTENTIAL RELEVANCE: The future athletic performance of Warmblood jumping horses may be limited as a result of OCD in the femoropatellar joint and to a certain extent the metacarpo/metatarsophalangeal joint. [less ▲]

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See detailEvaluation of acepromazine-induced hemodynamic alterations and reversal with norepinephrine infusion in standing horses
Pequito, Manuel; Amory, Hélène ULg; De Moffarts, Brieux et al

in Canadian Veterinary Journal = Revue Vétérinaire Canadienne (2013), 54

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See detailOsteochondosis in foals
Sandersen, Charlotte ULg; Vander Heyden, Laurent ULg; Detilleux, Johann ULg et al

in Veterinary Record : Journal of the British Veterinary Association (2013), 127(17), 456-467

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See detailCurcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils
Derochette, Sandrine ULg; Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg et al

in Chemico-Biological Interactions (2013), 206

In neutrophils (PMNs), superoxide anion (O2●-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation ... [more ▼]

In neutrophils (PMNs), superoxide anion (O2●-), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10-4 to 10-6 M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2●- production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10-5 M, while resveratrol appeared to be an O2●- scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production. [less ▲]

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See detailEffect of myeloperoxidase and anoxia/reoxygenation on mitochondrial respiratory function of cultured primary equine skeletal myoblasts.
Ceusters, Justine ULg; Mouithys-Mickalad, Ange ULg; Franck, Thierry ULg et al

in Mitochondrion (2013), 13(5),

Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction ... [more ▼]

Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function. [less ▲]

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See detailAssociation of breeding conditions with prevalence of osteochondrosis in foals
Vander Heyden, Laurent ULg; Lejeune, Jean-Philippe ULg; Caudron, Isabelle ULg et al

in Veterinary Record : Journal of the British Veterinary Association (2013), 178

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet ... [more ▼]

Osteochondrosis (OC) is the most common developmental orthopaedic disease in horses and represents a major problem to the horse industry. The complete mechanism of this multifactorial disease is not yet elucidated, but it is accepted that OC lesions are the result of intrinsic genetic and external factors. The aim of the present work was to evaluate the relationship between breeding management and OC. Breeding conditions were recorded, and a radiological examination was performed in 223 foals. Feeding practice and housing management were analysed in a multivariate model to determine risk factors for OC in three periods: gestation, birth to weaning and weaning to one-year-old. The major breakthrough of this study is the significant relationship between OC development and (1) the maternal nutrition during gestation and (2) the type of housing of the foals during their first year. It appears that mares fed with concentrates during gestation are more likely to produce foals that are subsequently affected by OC compared with other mares (P<0.05). Foals housed exclusively at pasture until one year of age are significantly less affected than foals exclusively housed in box or, alternatively, in box and at pasture (P<0.05). These results underline the role of the energy metabolism and the level of exercise in the aetiologic process of the disease, and help to develop preventive strategies during the crucial period of gestation to one year of age of the foal. [less ▲]

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See detailSevoflurane inhibits equine myeloperoxidase release and activity in vitro.
MINGUET, Grégory ULg; de la Rebière de Pouyade, Geoffroy ULg; Franck, Thierry ULg et al

in Veterinary Anaesthesia & Analgesia (2013), 40

Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs ... [more ▼]

Objective To investigate the effects of the volatile anaesthetic sevoflurane on the release of total and active myeloperoxidase (MPO) by non-stimulated and stimulated polymorphonuclear neutrophils (PMNs) in whole blood from healthy horses. Study design In vitro experimental study. Animals Adult healthy horses. Methods Samples of whole venous blood were collected and incubated in air or in air plus 2.3% or 4.6% sevoflurane for 1 hour. PMNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), with a combination of cytochalasin B (CB) and fMLP or with phorbol myristate acetate (PMA). Total and active MPO contents released by PMNs in blood were measured by enzyme-linked immunosorbent assay (ELISA) and specific immunological extraction followed by enzymatic detection (SIEFED) respectively. Additional experiments were performed to assess the effect of sevoflurane on the peroxidase and chlorination cycles of purified equine MPO using Amplex Red and 3'-(p-aminophenyl) fluorescein as fluorogenic substrates respectively. Results As compared with air alone, 1 hour exposure of whole blood to 4.6% sevoflurane in air significantly inhibited the release of total and active MPO by unstimulated and both fMLP- and CB + fMLP-stimulated PMNs but not by PMA-stimulated PMNs. Although 2.3% sevoflurane had no effect on total MPO release by unstimulated and stimulated PMNs, it significantly reduced the release of active MPO by unstimulated and fMLP-stimulated PMNs. Additionally, sevoflurane reversibly inhibited the activity of MPO, especially the peroxidase cycle of the enzyme. Conclusions and clinical relevance Although our experimental study was not designed to assess the effects of sevoflurane in vivo, this inhibition of MPO release and activity may have relevance for anaesthetized horses and deserves further studies to examine the clinical importance of these findings. [less ▲]

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See detailAltered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy
Tosi, Irène ULg; Art, Tatiana ULg; Cassart, Dominique ULg et al

Poster (2013)

Introduction: Exertional rhabdomyolyses are a common cause of exercise intolerance in the equine athlete, and Polysaccharide Storage Myopathy (PSSM) is a widely described muscular pathology. It is ... [more ▼]

Introduction: Exertional rhabdomyolyses are a common cause of exercise intolerance in the equine athlete, and Polysaccharide Storage Myopathy (PSSM) is a widely described muscular pathology. It is characterized by an accumulation of abnormal glycogen in myofibers due to a genetic defect in the skeletal muscle glycogen synthase (GYS1) enzyme. We hypothesized that the energetic production through the oxidative phosphorylation (OXPHOS) in muscular mitochondria might be impaired in type-1 PSSM-affected horses. Material and Methods: Eight horses with a history of exertional rhabdomyolysis were tested for the GYS1 mutation. Muscle biopsies were collected and used for histological analysis and high resolution respirometry (HRR). HRR values from 3 groups of horses (PSSM-positive horses, horses with a history of myopathy but negative to PSSM and healthy controls) were compared using a linear mixed model to take into account repeated (2-3 times) measurements made for each horse. Results: In four horses histology revealed an accumulation of abnormal glycogen in myofibers. A severe depression of the maximal OXPHOS capacity was observed by HRR in all horses with exertional rhabodmyolysis, with lower values in PSSM-positive cases. Conclusions: Our study shows a severe decreased OXPHOS capacity in PSSM-affected horses. PSSM is considered primarily a defect in glycogen synthesis but altered OXPHOS might play a central role in its pathogenesis. [less ▲]

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See detailTriphenylphosphonium salts of 1,2,4-benzothiadiazine 1,1-dioxides related to diazoxide targeting mitochondrial ATP-sensitive potassium channels
Constant-Urban, C.; Charif, M.; Goffin, Eric ULg et al

in Bioorganic & Medicinal Chemistry Letters (2013), 23

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See detailDifferentiation between stoichiometric and anticatalytic antioxidant properties of benzoic acid analogues: A structure/redox potential relationship study.
Franck, Thierry ULg; Mouithys-Mickalad, Ange ULg; Robert, Thierry ULg et al

in Chemico-biological interactions (2013), 206(2), 194-203

We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities ... [more ▼]

We investigated the antioxidant activities of some phenolic acid derivatives on a cell free system and on cellular and enzymatic models involved in inflammation. The stoichiometric antioxidant activities of phenolic acid derivatives were studied by measuring their capacity to scavenge the radical cation 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+) and reactive oxygen species (ROS) produced by stimulated neutrophils. The anticatalytic antioxidant capacity of the molecules was evaluated on the activity of myeloperoxidase (MPO), an oxidant enzyme present in and released by the primary granules of neutrophils. The ROS produced by PMA-stimulated neutrophils were measured by lucigenin-enhanced chemiluminescence (CL) and the potential interaction of the molecules with MPO was investigated without interferences due to medium by Specific Immuno-Extraction Followed by Enzyme Detection (SIEFED). The antioxidant activities of the phenolic compounds were correlated to their redox potentials measured by differential pulse voltammetry (DPV), and discussed in relation to their molecular structure. The ability of the phenolic molecules to scavenge ABTS radicals and ROS derived from neutrophils was inversely correlated to their increased redox potential. The number of hydroxyl groups (three) and their position (catechol) were essential for their efficacy as stoichiometric antioxidants or scavengers. On MPO activity, the inhibitory capacity of the molecules was not really correlated with their redox potential. Likewise, for the inhibition of MPO activity the number of OH groups and mainly the elongation of the carboxylic group were essential, probably by facilitating the interaction with the active site or the structure of the enzyme. The redox potential measurement, combined with ABTS and CL techniques, seems to be a good technique to select stoichiometric antioxidants but not anticatalytic ones, as seen for MPO, what rather involves a direct interaction with the enzyme. [less ▲]

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See detailEffects of diazoxide, benzothiadiazine and benzopyrane derivatives on mitochondrial proton and electron leaks of cardiomyocytes (H9C2 cell line).
Mouithys-Mickalad, Ange ULg; Ceusters, Justine ULg; Charef, M et al

Poster (2013)

Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of ... [more ▼]

Background: Mitochondria are double membrane- organelles that play a central role in cellular metabolism, calcium homeostasis and redox signaling. They have been also considered as main producers of adenosine triphosphate (ATP) and reactive oxygen species (ROS). In many cancer cells those organelles become dysfunctional leading to a shift of energy metabolism from oxidative phosphorylation to active glycolysis and an increase of ROS generation. According to Warberg’ theory, cancer damage might occur at the mitochondrial level, affecting tiny structures within each cell implicated in the energy production through ATP. New insight is that mitochondria might be a good therapeutic target for metabolic syndromes, ischemia/reperfusion injury and organs transplantation. Therefore, search for novel molecules able to keep mitochondria functional are of relevant interest. Methodology: Cardiomyocytes (H9C2 cells) were from ATCC (USA) and grown till confluence. The basal cellular respiratory rate, proton and electron leaks as well as ATP production were measured with the High Resolution Oxygraphy (Oroboros, Austria). All compounds: diazoxide (DIAZ), diazoxide –related analogs (1: BPDZ-259, 2: BPDZ-444), and benzopyran derivatives (3: BPDZ-490, 4: BPDZ-711) were tested at final concentration of 10-5 M, except when specified and compared to control samples (cells with or without DMSO). Results and conclusion: The basal respiratory rate of H9C2 cells (5x106/mL) was changed depending on the chemical structure of the tested compounds: e.g. compound 3 strongly enhanced the routine respiration, while 4 displayed a marked lowering effect. In contrast, the addition of similar concentration of benzothiadiazin derivatives (1, 2) had no effect on routine respiration but also on the other respiratory parameters such as oligomycin-induced leak and ATP production. Similar profile was obtained with the reference molecule: diazoxide. Overall, our findings indicate that both diazoxide-like analogues (1 and 2) and diazoxide were without significant effect on basal respiration, ATP production, even on maximal respiration. Interestingly, two derivatives show opposite effects: compound 3 behaves as a uncoupling agent and the other one (4) exhibits a real lowering effect on respiration but that was reversible. The latter effect might be of interest if this kind of molecules could be used for further use as an agent for organ conservation during transplantation. Our results also demonstrate that diazoxide, a well-known Mito-KATP opener, did not exert its effect beside of clinical situation like ischemia/reperfusion injury. [less ▲]

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See detailAn in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.
Ceusters, Justine ULg; Serteyn, Didier ULg; MINGUET, Grégory ULg et al

in Veterinary Immunology and Immunopathology (2012), 150(3-4), 221-7

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate ... [more ▼]

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-alpha in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-alpha, LPS and LPS/TNF-alpha, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-alpha significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity. [less ▲]

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See detailINFLUENCE OF MYELOPEROXIDASE ACTIVITY ON EQUINE POST-THAW SEMEN QUALITY
Ponthier, Jérôme ULg; Franck, Thierry ULg; Parrilla Hernandez, Sonia ULg et al

in Edeas, Marvin (Ed.) Proceedings of the 2nd ISANH World congress on Fertility and Antioxidants (2012, December 06)

This study confirms that active MPO is associated with cellular fraction of the ejaculate, as previously suggested for total MPO concentration in thawed semen (2). However, active MPO concentrations were ... [more ▼]

This study confirms that active MPO is associated with cellular fraction of the ejaculate, as previously suggested for total MPO concentration in thawed semen (2). However, active MPO concentrations were dramatically lower than total MPO concentrations observed in equine semen (3), which could be explained by presence of inactive MPO subunits in semen. MPO activity in sperm-rich pellet can be used as a predictive marker of post-thaw semen quality. Moreover, methods to inhibit MPO should be investigated in semen. [less ▲]

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