N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H(2)O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.Catalysis of hypochlorous acid production is the accepted physiological function of myeloperoxidase (MPO) 2 (1, 2). This heme enzyme is the major protein in neutrophils and is also present in monocytes, macrophages, microglia (3), and neurons (4, 5). It reacts with hydrogen peroxide to form the redox intermediate compound I, which oxidizes chloride to hypochlorous acid with coincident regeneration of the native enzyme (Reactions 1 and 2). HOCl indicates hypochlorous acid.Myeloperoxidase also promotes the oxidation of numerous substrates (RH) to free radical intermediates via the classical peroxidase cycle involving compound I and compound II (Reactions 3 and 4).Klebanoff (6) was the first to show that myeloperoxidase has potent antimicrobial activity because of its ability to generate hypochlorous acid. He proposed that myeloperoxidase produces hypochlorous acid inside phagosomes where it reacts with and kills ingested bacteria. However, during microbial killing myeloperoxidase functions in the presence of a high flux of superoxide (7). Superoxide reacts rapidly with native enzyme to produce oxymyeloperoxidase or compound III (Reaction 5; k 5 ϭ 2 ϫ 10 6 M Ϫ1 s Ϫ1 (8)), which is the dominant form of myeloperoxidase in stimulated neutrophils (9). Reactions of superoxide with myeloperoxidase are likely to be important in host defense because it has been demonstrated that superoxide enhances myeloperoxidasedependent killing of Staphylococcus aureus by isolated human neutrophils (10). The nature of this interaction has yet to be revealed.Compound II...

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Arthrospiraplatensis was cultivated in minitanks at 13 klux, using a mixture of KNO(3) and NH(4)Cl as nitrogen source. Fed-batch daily supply of NH(4)Cl at exponentially-increasing feeding rate allowed preventing ammonia toxicity and nitrogen deficiency, providing high maximum cell concentration (X(m)) and high-quality biomass (21.85 mg chlorophyll g cells(-1); 20.5% lipids; 49.8% proteins). A central composite design combined to response surface methodology was utilized to determine the relationships between responses (X(m), cell productivity and nitrogen-to-cell conversion factor) and independent variables (KNO(3) and NH(4)Cl concentrations). Under optimum conditions (15.5mM KNO(3); 14.1mM NH(4)Cl), X(m) was 4327 mg L(-1), a value almost coincident with that obtained with only 25.4mM KNO(3), but more than twice that obtained with 21.5mM NH(4)Cl. A 30%-reduction of culture medium cost can be estimated when compared to KNO(3)-batch runs, thus behaving as a cheap alternative for the commercial production of this cyanobacterium.

Sperandio PA, Oliveira MF, Rodrigues MK, Berton DC, Treptow E, Nery LE, Almeida DR, Neder JA. Sildenafil improves microvascular O 2 delivery-to-utilization matching and accelerates exercise O 2 uptake kinetics in chronic heart failure. Am J Physiol Heart Circ Physiol 303: H1474 -H1480, 2012. First published September 28, 2012; doi:10.1152/ajpheart.00435.2012 can temporally and spatially match microvascular oxygen (O 2) delivery (Q O2mv) to O2 uptake (V O2) in the skeletal muscle, a crucial adjustment-to-exercise tolerance that is impaired in chronic heart failure (CHF). To investigate the effects of NO bioavailability induced by sildenafil intake on muscle Q O2mv-to-O2 utilization matching and V O2 kinetics, 10 males with CHF (ejection fraction ϭ 27 Ϯ 6%) undertook constant work-rate exercise (70 -80% peak). Breath-bybreath V O2, fractional O2 extraction in the vastus lateralis {ϳdeoxy-genated hemoglobin ϩ myoglobin ([deoxy-Hb ϩ Mb]) by nearinfrared spectroscopy}, and cardiac output (CO) were evaluated after sildenafil (50 mg) or placebo. Sildenafil increased exercise tolerance compared with placebo by ϳ20%, an effect that was related to faster onand off-exercise V O2 kinetics (P Ͻ 0.05). Active treatment, however, failed to accelerate CO dynamics (P Ͼ 0.05). On-exercise [deoxy-Hb ϩ Mb] kinetics were slowed by sildenafil (ϳ25%), and a subsequent response "overshoot" (n ϭ 8) was significantly lessened or even abolished. In contrast, [deoxy-Hb ϩ Mb] recovery was faster with sildenafil (ϳ15%). Improvements in muscle oxygenation with sildenafil were related to faster on-exercise V O2 kinetics, blunted oscillations in ventilation (n ϭ 9), and greater exercise capacity (P Ͻ 0.05). Sildenafil intake enhanced intramuscular Q O2mv-to-V O2 matching with beneficial effects on V O2 kinetics and exercise tolerance in CHF. The lack of effect on CO suggests that improvement in blood flow to and within skeletal muscles underlies these effects.sildenafil; blood flow; heart failure; hemodynamics; near-infrared spectroscopy; oxygen consumption; kinetics THE INABILITY TO MAINTAIN an adequate driving pressure for blood-myocite oxygen (O 2 ) diffusion [i.e., microvascular partial pressure of O 2 (PO 2mv )] is paramount to explain the slowness of exercise O 2 uptake (V O 2 ) kinetics in patients with chronic heart failure [CHF; as recently reviewed by Poole and colleagues (34)]. To keep a sufficiently high PO 2mv , however, O 2 delivery should be spatially and temporally matched to V O 2 of individual fibers. In this context, seminal studies found that intramuscular PO 2mv in rodents with CHF was critically low either at rest-to-contractions transition (7,14) or during early recovery (12), i.e., when V O 2 should be increasing or decreasing most rapidly, respectively. Importantly, it was demonstrated that reduced nitric oxide (NO) bioavailability exerted a key mechanistic role on on-and off-exercise O 2 delivery-toutilization uncoupling in these animal preparations (24,25).In intact humans with CHF, previous studies have concomitant...

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