Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPOdependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces. (Blood. 2011; 117(4):1350-1358) IntroductionRecruitment of polymorphonuclear neutrophils (PMNs) is considered a hallmark in host defense. 1 With vessel-and tissue-infiltrating PMNs also being mechanistically linked to a broad range of vascular inflammatory diseases including coronary artery disease, heart failure and ischemia/reperfusion injury, the pathophysiologic significance of PMN motility reaches far beyond innate immunity. [2][3][4] So far, PMN migration is primarily viewed to be energydependent and cytoskeleton-dependent with G-protein coupled receptors and integrins initiating and orchestrating signaling pathways obligatory for neutrophil adhesion, spreading, diapedesis and chemotactic agitation. [5][6][7] On activation, PMN releases myeloperoxidase (MPO), an abundant heme protein in PMN with potent bactericidal and vascular-inflammatory properties. The enzyme accumulates along the endothelium and in the subendothelial space, 8 where it binds to anionic glycocalyx residues such as heparan sulfate glycosaminoglycans. Given the affinity of MPO to both PMN and endothelial cells, we evaluated whether MPO affects PMN locomotion and recruitment. Methods Isolation of PMNPeripheral blood was drawn from healthy human volunteers and heparinized, and isolation of PMN was performed as previously described. 9 In brief, after sedimentation in dextran solution (45 mg/mL), the supernatant was placed over Histopaque 1077 (Sigma-Aldrich) for density gradient centrifugation. Remaining red blood cells were eliminated by hypotonic lysis and the pellet was resuspended in Hanks balanced salt solution (HBSS; Invitrogen) containing 0...

Our data indicate that UVA irradiation induces UPE especially in deeper (living) skin layers, where antioxidants must be active in order to interfere with accelerated skin ageing. Based on the clinical data, and with knowledge of modulating external variables, UPE measurement following UV excitation can be qualified as a reliable and valid method for the non-invasive measurement of antioxidant efficacy on the skin.

Measurement of UPE could be proven to be a highly sensitive method to assess oxidative processes in biological molecules. The reported data indicate that UPE generated by oxidation stressed skin is mainly due to non-fluorescent photon emission via Trp, whereby Trp acts as an energy receptor from other excited species of oxidation-modified amino acids.

Our previous results showed that inhibition of protein tyrosine phosphatases (PTP) by orthovanadate is an appropriate strategy to mimic nerve growth factor (NGF) effects in neurons, including enhanced phosphorylation of TrkA, stimulation of downstream survival signaling pathways, and protection against apoptotic stress. In this study, we wanted to trigger such NGF-like survival signaling in primary hippocampal neurons with the more specific PTP inhibitors ethyl-3,4-dephostatin (DPN), 4-O-methyl-ethyl-3,4-dephostatin (Me-DPN), and methoxime-3,4-dephostatin. It was striking that only the nitric oxide (NO)-releasing dephostatin analogs DPN and Me-DPN, but not the nitrosamine-free methoxime derivative (which did not release NO), enhanced TrkA phosphorylation and protected the neurons against staurosporine (STS)-induced apoptosis. The established NO donor S-nitroso-N-acetylpenicillamine (SNAP) also enhanced TrkA phosphorylation and prevented apoptosis similarly to DPN and Me-DPN. Analysis of the major signaling pathways downstream of TrkA revealed that both SNAP and DPN enhanced phosphorylation of Akt and the mitogen-activated kinases (MAPK) Erk1/2. Blocking of these signaling pathways by the PI3-K inhibitor wortmannin or the MAPK kinase inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] equally abolished the neuroprotective effect of the NO donors. It was striking that inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or protein kinase G (PKG) inhibition by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg: 3Ј,2Ј,1Ј-kl]pyrrolo [3,4-i] [1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823) also blocked the neuroprotective effect of the NO donors, and ODQ clearly attenuated SNAPinduced phosphorylation of TrkA, Akt, and MAPK. In conclusion, NO release by the dephostatin derivatives and subsequent stimulation of sGC and PKG is essential for their neuroprotective effects. In primary neurons, such NO-activated survival signaling involves NGF-like effects, including enhanced phosphorylation of TrkA and activation of PI3-K/Akt and MAPK pathways.Nitric oxide is an endogenous regulator of different cellular functions, including vascular tone (Gruetter et al., 1979;Furchgott and Zawadzki, 1980), neurotransmission (Vincent, 1994;Garthwaite and Boulton, 1995), inflammation (Nathan and Shiloh, 2000), and cellular signaling cascades (Dawson et al., 1992). In neurons, NO has been found to be a Janus-faced molecule that can mediate survival signaling (Troy et al., 2000;Contestabile and Ciani, 2004) but may on the other hand contribute to neuronal death and brain damage in neurological diseases [e.g., stroke (Zhang et al., 1996), Alzheimer's disease , amyotrophic lateral sclerosis (Urushitani and Shimohama, 2001), or Parkinson's disease (Liberatore et al., 1999)]. In the brain, NO can be Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.