GLUTATHIONE PEROXIDASE IN LENS AND A SOURCE OF HYDROGEN PEROXIDE IN AQUEOUS HUMOUR.
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ABSTRACT:
SUBMITTER: PIRIE A
PROVIDER: S-EPMC1206928 | biostudies-other | 1965 Jul
REPOSITORIES: biostudies-other
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Project description:AIMS:Ero1 flavoproteins catalyze oxidative folding in the endoplasmic reticulum (ER), consuming oxygen and generating hydrogen peroxide (H2O2). The ER-localized glutathione peroxidase 7 (GPx7) shows protein disulfide isomerase (PDI)-dependent peroxidase activity in vitro. Our work aims at identifying the physiological role of GPx7 in the Ero1?/PDI oxidative folding pathway and at dissecting the reaction mechanisms of GPx7. RESULTS:Our data show that GPx7 can utilize Ero1?-produced H2O2 to accelerate oxidative folding of substrates both in vitro and in vivo. H2O2 oxidizes Cys57 of GPx7 to sulfenic acid, which can be resolved by Cys86 to form an intramolecular disulfide bond. Both the disulfide form and sulfenic acid form of GPx7 can oxidize PDI for catalyzing oxidative folding. GPx7 prefers to interact with the a domain of PDI, and intramolecular cooperation between the two redox-active sites of PDI increases the activity of the Ero1?/GPx7/PDI triad. INNOVATION:Our in vitro and in vivo evidence provides mechanistic insights into how cells consume potentially harmful H2O2 while optimizing oxidative protein folding via the Ero1?/GPx7/PDI triad. Cys57 can promote PDI oxidation in two ways, and Cys86 emerges as a novel noncanonical resolving cysteine. CONCLUSION:GPx7 promotes oxidative protein folding, directly utilizing Ero1?-generated H2O2 in the early secretory compartment. Thus, the Ero1?/GPx7/PDI triad generates two disulfide bonds and two H2O molecules at the expense of a single O2 molecule.
| S-EPMC3901321 | biostudies-literature
Project description:We show H2O2 is spontaneously produced from pure water by atomizing bulk water into microdroplets (1 ?m to 20 µm in diameter). Production of H2O2, as assayed by H2O2-sensitve fluorescence dye peroxyfluor-1, increased with decreasing microdroplet size. Cleavage of 4-carboxyphenylboronic acid and conversion of phenylboronic acid to phenols in microdroplets further confirmed the generation of H2O2 The generated H2O2 concentration was ?30 µM (?1 part per million) as determined by titration with potassium titanium oxalate. Changing the spray gas to O2 or bubbling O2 decreased the yield of H2O2 in microdroplets, indicating that pure water microdroplets directly generate H2O2 without help from O2 either in air surrounding the droplet or dissolved in water. We consider various possible mechanisms for H2O2 formation and report a number of different experiments exploring this issue. We suggest that hydroxyl radical (OH) recombination is the most likely source, in which OH is generated by loss of an electron from OH- at or near the surface of the water microdroplet. This catalyst-free and voltage-free H2O2 production method provides innovative opportunities for green production of hydrogen peroxide.
| S-EPMC6765303 | biostudies-literature
Project description:The organocatalytic epoxidation of unactivated alkenes using aqueous hydrogen peroxide provides various indispensable products and intermediates in a sustainable manner. While formyl functionalities typically undergo irreversible oxidations when activating an oxidant, an atropisomeric two-axis aldehyde capable of catalytic turnover was identified for high-yielding epoxidations of cyclic and acyclic alkenes. The relative configuration of the stereogenic axes of the catalyst and the resulting proximity of the aldehyde and backbone residues resulted in high catalytic efficiencies. Mechanistic studies support a non-radical alkene oxidation by an aldehyde-derived dioxirane intermediate generated from hydrogen peroxide through the Payne and Criegee intermediates.
| S-EPMC8336450 | biostudies-literature
Project description:PurposeTo compare aqueous humour (AH) dynamics in the presence of a precrystalline (Implantable Collamer Lens®; ICL) or iris-fixed (Artiflex®) phakic intraocular lens (PIOL).MethodsBy computational fluid dynamics simulation, AH flow was modelled through a peripheral iridotomy (PI) or central lens hole (both 360 ?m) in the presence of an Artiflex or ICL lens, respectively. The impacts of AH flow were then determined in terms of wall shear stress (WSS) produced on the endothelium or crystalline lens. Effects were also modelled for different scenarios of pupil diameter (PD 3.5 or 5.5 mm), ICL vault (100, 350, 800 ?m) and number of Artiflex iridotomies (1 or 2) and location (12 or 6 o'clock).ResultsFor a PD of 3.5 mm, AH volumes flowing from the posterior to the anterior chamber were 37.6% of total flow through the lens hole (ICL) and 84.2% through PI (Artiflex). For an enlarged PD (5.5 mm), corresponding values were 10.3% and 81.9% respectively, so PI constitutes a very efficient way of evacuating AH. Central endothelial WSS in Pa was lower for the large vault ICL and the Artiflex (1-03 and 1.1-03 respectively) compared to the PIOL-free eye (1.6-03). Crystalline lens WSS was highest for the lowest vault ICL (1-04).ConclusionsAH flow varied according to the presence of a precrystalline or iris-fixed intraocular lens. Endothelial WSS was lower for an implanted ICL with large vault and Artiflex than in the PIOL-free eye, while highest crystalline WSS was recorded for the lowest vault ICL.
| S-EPMC6089426 | biostudies-literature
Project description:This work demonstrates that hydrogen peroxide (H2O2) is generated in weak polyacrylamide hydrogels due to mechanochemical reactions to osmotic swelling. Hydrogels are important tools and materials for many biomedical applications, particularly for growth of stem cells. However, swollen gels are under constant tension, which makes their individual chains susceptible to mechanochemical bond breakage. In this work, an assay was developed to measure the generation of H2O2 as a result of hydrogel swelling. Polyacrylamide hydrogels with both weak disulfide and strong PEG-diacrylate crosslinkers were synthesized and swelled. H2O2 generation increased in the presence of weaker crosslinkers, up to 30 ?M H2O2, whereas stronger crosslinkers reduced this to 5 ?M H2O2. H2O2 levels decreased when swelled in the presence of dextran to reduce osmotic stress or increased if the gels were conjugated to an acrylated surface. Finally, H2O2 continued to form for days after the gels had reached their equilibrium sizes, independently of dissolved oxygen. The results of this work impact those working in the 3D cell culture community and demonstrate that even well-characterized systems undergo mechanochemical processes in mild environments.
| S-EPMC6687575 | biostudies-literature
Project description:High levels of reactive oxygen species such as hydrogen peroxide (H2O2) cause oxidative stress in the lens and lead to cataractogenesis. The present investigation was undertaken to find out whether the mammalian lens aquaporins (AQPs) 0, 1, and 5 perform H2O2 transport across the plasma membrane to reduce oxidative stress. Our in vitro cell culture and ex vivo lens experiments demonstrated that in addition to the established water transport role, mouse AQP0, AQP1 and AQP5 facilitate transmembrane H2O2 transport and function as peroxiporins. Human lens epithelial cells expressing AQP1, AQP5 and AQP8, when treated with 50 μM HgCl2 water channel inhibitor showed a significant reduction in H2O2 transport. Data obtained from the experiments involving H2O2-degrading enzyme glutathione peroxidase 1 (GPX1) knockout lenses showed H2O2 accumulation, suggesting H2O2 transport level by AQPs in the lens is regulated by GPX1. Under hyperglycemic conditions, there was an increased loss of transparency, and enhanced production and retention of H2O2 in AQP5-/- lenses compared to similarly-treated WT lenses. Overall, the results show that lens AQPs function as peroxiporins and cooperate with GPX1 to maintain lens H2O2 homeostasis to prevent oxidative stress, highlighting AQPs and GPX1 as promising therapeutic drug targets to delay/treat/prevent age-related lens cataracts.
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Project description:Neutrophils must navigate accurately towards pathogens in order to destroy invaders and thus defend our bodies against infection. Here we show that hydrogen peroxide, a potent neutrophil chemoattractant, guides chemotaxis by activating calcium-permeable TRPM2 ion channels and generating an intracellular leading-edge calcium "pulse". The thermal sensitivity of TRPM2 activation means that chemotaxis towards hydrogen peroxide is strongly promoted by small temperature elevations, suggesting that an important function of fever may be to enhance neutrophil chemotaxis by facilitating calcium influx through TRPM2. Chemotaxis towards conventional chemoattractants such as LPS, CXCL2 and C5a does not depend on TRPM2 but is driven in a similar way by leading-edge calcium pulses. Other proposed initiators of neutrophil movement, such as PI3K, Rac and lyn, influence chemotaxis by modulating the amplitude of calcium pulses. We propose that intracellular leading-edge calcium pulses are universal drivers of the motile machinery involved in neutrophil chemotaxis.
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Project description:Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O₂ consumed as H₂O₂ upon addition of NADPH. Addition of substrate had little effect on H₂O₂ production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H₂O₂. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H₂O₂, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H₂O₂ generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H₂O₂ release. Catalase did not reduce levels of H₂O₂ with either FMO2.1 or FMO3 but inhibited H₂O₂ generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H₂O₂.
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Project description:The activity of ascorbate peroxidase (APX) has been studied with H(2)O(2) and various reducing substrates. The activity decreased in the order pyrogallol>ascorbate>guaiacol>2, 2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The inactivation of APX with H(2)O(2) as the sole substrate was studied. The number of H(2)O(2) molecules required for maximal inactivation of the enzyme was determined as approx. 2.5. Enzymic activity of approx. 20% of the original remained at the end of the inactivation process (i.e. approx. 20% resistance) when ascorbate or ABTS was used as the substrate in activity assays. With pyrogallol or guaiacol no resistance was seen. Inactivation by H(2)O(2) followed over time with ascorbate or pyrogallol assays exhibited single-exponential decreases in enzymic activity. Hyperbolic saturation kinetics were observed in both assay systems; a similar dissociation constant (0.8 microM) for H(2)O(2) was obtained in each case. However, the maximum rate constant (lambda(max)) obtained from the plots differed depending on the assay substrate. The presence of reducing substrate in addition to H(2)O(2) partly or completely protected the enzyme from inactivation, depending on how many molar equivalents of reducing substrate were added. An oxygen electrode system has been used to confirm that APX does not exhibit a catalase-like oxygen-releasing reaction. A kinetic model was developed to interpret the experimental results; both the results and the model are compared and contrasted with previously obtained results for horseradish peroxidase C. The kinetic model has led us to the conclusion that the inactivation of APX by H(2)O(2) represents an unusual situation in which no enzyme turnover occurs but there is a partition of the enzyme between two forms, one inactive and the other with activity towards reducing substrates such as ascorbate and ABTS only. The partition ratio is less than 1.
| S-EPMC1221069 | biostudies-other
Project description:Key pointsAn elevation in intracranial pressure (ICP) lowers conventional outflow facility (increases aqueous outflow resistance) of rat eyes. The reduction in outflow facility correlates with an increase in intraocular pressure (IOP). The effect of ICP elevation on outflow facility and IOP is blocked by TTX. The results indicate that aqueous humour dynamics is modulated by ICP-driven neural feedback from the brain. This feedback mechanism may act to stabilize translaminar pressure across the optic nerve head and may provide a new avenue for glaucoma therapy.AbstractWhile intraocular pressure (IOP) is a well-known risk factor for glaucoma, intracranial pressure (ICP) is attracting heightened interest because of its influence on optic nerve head biomechanics. Studies have shown that ICP can have marked impacts on posterior eye health by modifying the translaminar pressure gradient across the optic nerve. There is also growing evidence that IOP and ICP may be interconnected, although the mechanism of their putative interaction is unknown. We sought to test the hypothesis that ICP modulates IOP by altering aqueous humour dynamics. The anterior chamber and lateral ventricle of anaesthetized Brown-Norway rats were cannulated with fine-gauge needles connected to a programmable pump and saline reservoir, respectively. ICP was manipulated by varying reservoir height, and eye outflow facility (C) was determined from the pump flow rate required to hold IOP at different levels. C was 22 ± 4 nl/min/mmHg at resting ICP and 13 ± 3 nl/min/mmHg when ICP was raised 15 mmHg, a reduction of 41 ± 13% (n = 18). The decrease in outflow facility was independent of blood pressure, reversible, scaled with ICP elevation and correlated with increases in resting IOP. It was physiological in origin because C returned to baseline values after the rats were killed and corneal application of TTX though ICP remained elevated. These results indicate that a neural feedback mechanism driven by ICP regulates conventional outflow facility in rats. The mechanism may protect the eye from translaminar pressure swings and may offer a new target for glaucoma treatment.
| S-EPMC7018442 | biostudies-literature