Ferric human neuroglobin scavenges superoxide to form oxy adduct.

Neuroglobin (Ngb) is the third member of the vertebrate globin family, and the structure was solved as a typical globin fold with a b-type heme. Although it has been proposed that Ngb could be involved in neuroprotection against oxidative stress, the protective mechanism has not been fully identified yet. In order to clarify functions under hypoxic condition, in this study, we focused on the scavenger activity of human Ngb (hNgb) against superoxide. The activity of hNgb for superoxide was evaluated to be 7.4 µM for IC50, the half maximal inhibitory concentration. The result indicates that hNgb can be an anti-oxidant, and the value was almost the same as that of ascorbic acid. In addition, we characterized oxidation states of a heme iron in superoxide-treated hNgb with spectroscopic measurements. Superoxide-treated hNgb in the ferric form was readily converted to the oxygenated ferrous form, and the result suggested that ferric hNgb could scavenge superoxide by change of an oxidation state in a heme iron. Moreover, mutational experiments were performed, and the each variant mutated at 46 and 55 positions suggested a disulfide bond between Cys46 and Cys55 could be essential to be sensors for oxidative stress with the direct binding of superoxide. As a consequence, we concluded that redox changes of the heme iron and the disulfide bond could regulate neuroprotective functions of hNgb, and it suggests that hNgb can afford protection against hypoxic and ischemic stress in the brain.

Animal diseases caused by orbiviruses, Algeria.

Antibodies against bluetongue virus were detected in cattle, sheep, goats, and camels in Algeria in 2008. Antibodies against epizootic hemorrhagic disease virus were detected in cattle, but antibodies against African horse sickness virus were not detected in horses and mules. Epizootic hemorrhagic disease in northern Africa poses a major risk for the European Union.

Design of a practical fluorescent probe for superoxide based on protection-deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups.

A strategy for designing probes based on protection-deprotection chemistry involving fluoresceins and their benzenesulfonyl (BES) derivatives has led to the development of a much more practical superoxide (O(2) (-.)) probe than the previously reported bis(2,4-dinitro-BES) tetrafluorofluorescein (6 a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6 a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7 j) as the optimal reagent. A microtiter plate assay with BESSo showed a tenfold improved detection limit for O(2) (-.) compared with such an assay based on 6 a. BESSo showed markedly better specificity for O(2) (-.) than for GSH or other reactive oxygen species, and this specificity was significantly higher than that of Fe(2+) and some reducing enzymes. These features have resulted in the development of an assay based on BESSo that is capable of providing more unambiguous results for O(2) (-.) release from neutrophils, with or without stimulation by phorbol myristate acetate, as compared with an assay based on 6 a. Intracellular generation of O(2) (-.) in human Jurkat T cells stimulated by butyric acid has been measured by using flow cytometry and fluorescence microscopy utilizing the acetoxymethyl derivative of BESSo.

A design of fluorescent probes for superoxide based on a nonredox mechanism.

Fluorometric detection of O2-* is performed based on desulfonylation of 3 to the corresponding fluoresceins 4 through nucleophilic substitution, and this fluorescing process is quite specific toward O2-* over H2O2, t-BuOOH, NaOCl, 1O2, HO*, NO*, and ONOO-. Furthermore, effects of glutathione, cytochrome P450 reductase/NADPH, and diaphorase/NADH are relatively small on the fluorescing process of probe 3 with X = Y = F, which is useful to detect O2-* released from neutrophils stimulated by phorbol myristate acetate with satisfactory sensitivity.