Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells

Abstract In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22phox, but not Nox2 or p47phox, are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2/ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs. Antioxid. Redox Signal. 11, 747-764.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78121/1/ars.2008.2203.pd

A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B

We have proposed that the "doublet" EPR spectra observed during catalysis by a number of coenzyme B1,2-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B1,2r. By varying the magnitude of the exchange coupling we have quite accurately simulateed the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammonia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22002/1/0000415.pd

Harmful Waste Products as Novel Immune Modulators for Treating Inflammatory Arthritis?

Cope discusses a new study in rats suggesting that oxidative burst inducers might have a role to play in treating inflammatory arthritis

MutT from the fish pathogen Aliivibrio salmonicida is a cold active nucleotide pool sanitization enzyme with an unexpected high thermostability

AbstractUpon infection by pathogenic bacteria, production of reactive oxygen species (ROS) is part of the host organism’s first line of defence. ROS damage a number of macromolecules, and in order to withstand such a harsh environment, the bacteria need to have well-functioning ROS scavenging and repair systems. Herein, MutT is an important nucleotide-pool sanitization enzyme, which degrades 8-oxo-dGTP and thus prevents it from being incorporated into DNA. In this context, we have performed a comparative biochemical and structural analysis of MutT from the fish pathogen Aliivibrio salmonicida (AsMutT) and the human pathogen Vibrio cholerae (VcMutT), in order to analyse their function as nucleotide sanitization enzymes and also determine possible cold-adapted properties of AsMutT. The biochemical characterisation revealed that both enzymes possess activity towards the 8-oxo-dGTP substrate, and that AsMutT has a higher catalytic efficiency than VcMutT at all temperatures studied. Calculations based on the biochemical data also revealed a lower activation energy (Ea) for AsMutT compared to VcMutT, and differential scanning calorimetry experiments showed that AsMutT displayed an unexpected higher melting temperature (Tm) value than VcMutT. A comparative analysis of the crystal structure of VcMutT, determined to 2.42Å resolution, and homology models of AsMutT indicate that three unique Gly residues in loops of VcMutT, and additional long range ion-pairs in AsMutT could explain the difference in temperature stability of the two enzymes. We conclude that AsMutT is a stable, cold-active enzyme with high catalytic efficiency and reduced Ea, compared to the mesophilic VcMutT

Functional Defect in Neutrophil Cytosols from Two Patients with Autosomal Recessive Cytochrome-positive Chronic Granulomatous Disease

Abstract The kinetics of activation of the respiratory burst oxidase in the cell-free oxidase-activating system have been explained by a three-stage mechanism in which the membrane-associated oxidase components M: (a) take up a cytosolic factor S to form a complex M S that is (b) 48-kD proteins that are missing in certain forms of CGD, and that other forms of type II CGD besides the one described in this report remain to be discovered

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples

Leukotriene B4 mediates p47phox phosphorylation and membrane translocation in polyunsaturated fatty acidâ stimulated neutrophils

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142119/1/jlb0976.pd