Deconjugation Kinetics of Glucuronidated Phase II Flavonoid Metabolites by B-glucuronidase from Neutrophils

Flavonoids are inactivated by phase II metabolism and occur in the body as glucuronides. Mammalian ß-glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid glucuronides. We have studied deconjugation kinetics and pH optimum for four sources of ß-glucuronidase (human neutrophil, human recombinant, myeloid PLB-985 cells, Helix pomatia) with five flavonoid glucuronides (quercetin-3-glucuronide, quercetin-3'-glucuronide, quercetin-4'-glucuronide, quercetin-7-glucuronide, 3'-methylquercetin-3-glucuronide), 4-methylumbelliferyl-ß-D-glucuronide, and para-nitrophenol-glucuronide. All substrate-enzyme combinations tested exhibited first order kinetics. The optimum pH for hydrolysis was between 3.5-5, with appreciable hydrolysis activities up to pH 5.5. At pH 4, the Km ranged 44-fold from 22 µM for quercetin-4'-glucuronide with Helix pomatia ß-glucuronidase, to 981 µM for para-nitrophenol-glucuronide with recombinant ß-glucuronidase. Vmax (range: 0.735-24.012 µmol·min-1·unit-1 [1 unit is defined as the release of 1 µM 4-methylumbelliferyl-ß-D-glucuronide per min]) and the reaction rate constants at low substrate concentrations (k) (range: 0.002-0.062 min-1·(unit/L)-1 were similar for all substrates-enzyme combinations tested. In conclusion, we show that ß-glucuronidase from four different sources, including human neutrophils, is able to deconjugate flavonoid glucuronides and non-flavonoid substrates at fairly similar kinetic rates. At inflammatory sites in vivo the pH, neutrophil and flavonoid glucuronide concentrations seem favorable for deconjugation. However, it remains to be confirmed whether this is actually the case

Bedreigen virussen de palingpopulatie?

Resultaten van een onderzoek naar het voorkomen van virussen (EVEX, Eel Virus European X; HVA, Herpes Virus Anguillea; EVE, Eel Virus European) bij palingen afkomstig uit diverse landen (zowel wilde paling als paling van kwekerijen) en van een zwemexperiment met geïnfecteerde en niet-geïnfecteerde palingen. Na voltooiing van de zwemreis werd in beide groepen het bloedbeeld onderzocht op diverse parameters (o.a. op hematocriet, het aantal rode bloedcellen). De resultaten zijn zeer verontrustend, omdat ze een aanwijzing vormen dat virus-infectie ertoe leidt dat palingen hun migratie naar de paaigronden niet kunnen voltooien door bloedarmoede, een beschadigde lever en een tekort aan eiwitten. Dit kan mede een oorzaak zijn voor het wereldwijd teruglopen van de palingstan

An Essential Difference between the Flavonoids MonoHER and Quercetin in Their Interplay with the Endogenous Antioxidant Network

Antioxidants can scavenge highly reactive radicals. As a result the antioxidants are converted into oxidation products that might cause damage to vital cellular components. To prevent this damage, the human body possesses an intricate network of antioxidants that pass over the reactivity from one antioxidant to another in a controlled way. The aim of the present study was to investigate how the semi-synthetic flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside (monoHER), a potential protective agent against doxorubicin-induced cardiotoxicity, fits into this antioxidant network. This position was compared with that of the well-known flavonoid quercetin. The present study shows that the oxidation products of both monoHER and quercetin are reactive towards thiol groups of both GSH and proteins. However, in human blood plasma, oxidized quercetin easily reacts with protein thiols, whereas oxidized monoHER does not react with plasma protein thiols. Our results indicate that this can be explained by the presence of ascorbate in plasma; ascorbate is able to reduce oxidized monoHER to the parent compound monoHER before oxidized monoHER can react with thiols. This is a major difference with oxidized quercetin that preferentially reacts with thiols rather than ascorbate. The difference in selectivity between monoHER and quercetin originates from an intrinsic difference in the chemical nature of their oxidation products, which was corroborated by molecular quantum chemical calculations. These findings point towards an essential difference between structurally closely related flavonoids in their interplay with the endogenous antioxidant network. The advantage of monoHER is that it can safely channel the reactivity of radicals into the antioxidant network where the reactivity is completely neutralized

Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants

Introduction: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-L-cysteine (NAC) or glutathione (GSH). Methods: BEAS-2B cells were exposed for 24 h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63(+)CD81(+) bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. Results: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. Summary/conclusion: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein-and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment

Negative magnetoresistance in boron-doped nanocrystalline diamond films

We report on the observation of a negative magnetoresistance (NMR) regime in boron-doped nanocrystalline diamond films at low temperatures. A comparative analysis of our experimental results and those reported for systems composed of superconducting granules embedded in an insulating matrix (also referred as granular films) suggest the presence of superconducting regions inside the insulating films as causing the NMR. By considering the latter scenario, the experimental observations are explained by modeling the systems as consisting of a distribution of superconducting granules whose global properties are tuned by the intergrain distance

Bacterial diseases of tilapia, their zoonotic potential and risk of antimicrobial resistance

Tilapia culture is an important source of income and nutrition to many rural families. Since 2000, the production of tilapia increased and reached domestic and global markets. Major farmed species is Nile tilapia (Oreochromis niloticus), in earthen ponds and cage cultures. Intensification contributed to global tilapia disease outbreaks, with bacterial infections causing mortalities and morbidities, threatening sustainable production. At tilapia farms, high nutrient concentrations, water temperature and fish densities enhance bacterial growth including virulent bacterial clones and potential zoonotic bacteria. Global warming favours this. This review respectively provides a comprehensive overview of the most common and emerging bacterial pathogens, diseases, clinical presentations and diagnostics of tilapia, including bacteria and diseases with zoonotic potential. First, common bacterial disease outbreaks, including streptococcosis, motile Aeromonas septicaemia, francisellosis, columnaris disease and vibriosis are described. Then, information on emerging bacterial infections of concern for tilapia, like edwardsiellosis through Edwardsiella ictaluri and E. tarda, as well as Aeromonas schubertii is provided. Reports of infectious bacterial tilapia disease outbreaks from other bacteria, including Lactococcus garvieae, Aerococcus viridans, Pseudomonas spp., Mycobacterium marinum and Chlamydia spp., and others are reviewed. Furthermore, bacteria with zoonotic potential, like Streptococcus agalactiae ST283, S. iniae, Aeromonas sp., E. tarda, Vibrio vulnificus pathovar (pv) piscis and M. marinum are included in the review, to provide the most current overview of the disease risks affecting production and post-harvest stages. Additionally, the status and risks of antimicrobial resistance in bacteria from tilapia and other cultured fish through imprudent use of antibiotics, and its future at a global level are provided