Bernard M. Babior

Evidence for hydroxyl radical production by human neutrophils.

The possibility that neutrophils produce the hydroxyl radical (OH-) was studied by examining the ability of these cells to support the release of ethylene from methional, a reaction in which it has been shown that OH-, but not O2- or H2O2, may serve as the oxidizing agent. When neutrophils were exposed to opsonized zymosan in the presence of 0.35 mM methional, ethylene was released in quantities amounting to 44.6+/-3.6 pmol/10(6) cells/40 min. Ethylene production required the presence of neutrophils, opsonized zymosan, and methional, indicating that it was formed from methional by stimulated but not resting neutrophils. Ethylene was not produced by zymosan-treated cells from patients with chronic granulomatous disease, confirming the requirement for respiratory burst activity in this process. Ethylene production was suppressed by benzoic acid, an OH- scavenger. Superoxide dismutase (3 microgram/ml) reduced ethylene production to 21% of control levels, but catalase had no significant effect in this system. These findings indicate that stimulated neutrophils produce a highly reactive oxidizing radical, possibly OH-, which releases ethylene from methional, and that the O2-generated during the respiratory burst is involved in the production of this reactive species.

Neutrophil oxygen reduction: The enzymes and the products

Abstract The human neutrophil generates a non-mitochondrial respiratory burst by the activation of a NADPH-oxidase, whose electron source, NADPH, is generated in the hexose monophosphate shunt. The reduction product, 2−, is further reduced to H2O2, which upon the action of myeloperoxidase, oxidizes halide to form reactive chloramines and hypochlorous acid. The elusive hydroxyl radical, or kindred species, also appears as a product of the burst, but this chemistry has not been elucidated. NADPH-oxidase is a complex activity, comprised of at least two components: a low potential b cytochrome and a flavoprotein. Partial characterization and isolation of this electron transport system has been accomplished and serves as an intense focus of current research. The recent demonstration that the oxidase may be activated in a broken cell preparation should not only define mechanisms of burst activation, but this methodology should provide a powerful approach towards identifying the components of the NADPH-oxidase apparatus.

Unique characteristics of superoxide production by human eosinophils in eosinophilic states.

Eosinophils from patients with peripheral blood eosinophilia and human neutrophils from normal subjects and patients with neutrophilia produced superoxide anion (O2−) in vitro at similar rates in the absence of stimulation and exhibited comparably increased rates of O2− production during the initial 1 h of incubation with opsonized zymosan. In the presence of opsonized zymosan, the rate of O2− production by eosinophils was constantly high for 3 h, whereas the rate of production by neutrophils fell by more than 65% after 1 h. Consequently, the amount of superoxide produced by phagocytizing leukocytes was twofold higher for eosinophils than for neutrophils at 3 h. O2− production by cell-free sonicates of zymosan-stimulated eosinophils and neutrophils exhibited the same preference for NADPH over NADH. One mM sodium azide significantly decreased the generation of O2− by phagocytizing eosinophils, but lacked an effect on neutrophils. The prolonged release of O2− by eosinophils engaged in phagocytosis may contribute both to their unique microbicidal profile and to the capacity of eosinophils to injure host tissues in some eosinophilic syndromes.

O2- and host defense: the production and fate of O2- in neutrophils.

Abstract— The neutrophil is a circulating cell whose function is to find, ingest and destroy invading microorganisms. Among the weapons used by this cell against its target is a series of powerful oxidizing agents produced by the partial reduction of oxygen. Generation of these oxidizing agents is initiated when the neutrophil encounters its target. This encounter activates a flavoenzyme, dormant in resting cells, which catalyzes the reduction of oxygen to O2‐ using NADPH as the electron donor. The dismutation of O2‐ either spontaneously or under catalysis by superoxide dismutase, then gives rise to H2O2, a compound which is used in combination with Cl‐ and myeloperoxidase to provide an exceedingly powerful antimicrobial system. O2‐ also serves as a precursor of OH, another powerful oxidant which may be employed by the neutrophil as an antimicrobial agent. Singlet oxygen may also be formed in neutrophils.

Superoxide production by phagocytes. Another look at the effect of cytochrome c on oxygen uptake by stimulated neutrophils.

Abstract The widely held view that stimulated phagocytes liberate O 2 − into the extracellular medium is supported by the alterations in oxygen uptake which occur when ferricytochrome c is added to a suspension of zymosan-treated neutrophils. An explanation consistent with this view is provided for some previously reported results ( FEBS Lett . 100 , 27) which initially appeared to conflict with the notion that O 2 − is released by phagocytes.

Cytotoxins against a granulocyte antigen system: detection by a new method employing cytochalasin-B-treated cells.

Abstract. A method for demonstrating granulocyte cytotoxins using a modified microdroplet dye exclusion technique and cytochalasin‐B‐treated cells is described. Cytochalasin‐B‐treated granulocytes were reacted against 223 sera from multitransfused pregnant and renal transplant patients. Incidence of granulocyte cytotoxins was 11.2%. Analysis (2times2 contingency tables) of the reactivity of GCT + sera showed highly significant (p<0.001) positive associations among 5 sera and a significant (p<0.001) negative association with another serum. The data are consistent with the hypothesis that a granulocyte antigen system is detected by cytotoxins and that granulocyte antigens ‘Gr1’ and ‘Gr2’ may be products of allelic genes.

The mechanism of cobalamin-dependent rearrangements

SummaryAdenosylcobalamin-dependent rearrangements are enzyme catalyzed reactions in which a hydrogen atom is transferred from one carbon atom to an adjacent one in exchange for a group X which migrates in the opposite direction. In the hydrogen transfer step, the mechanism of which is reasonably well understood, the cofactor serves as an intermediate hydrogen carrier. The transfer of hydrogen to the cofactor involves homolysis of the carbon-cobalt bond to generate cob(II)alamin and the 5′-deoxyadenos-5′-yl radical, followed by abstraction of a hydrogen atom from the substrate to form 5′-deoxyadenosine and the substrate radical. After migration of group X, the hydrogen atom is returned to the product radical by the reverse of the above reactions to generate the final product and reconstitute the cofactor.In contrast to the transfer of hydrogen, the mechanism of group X migration is poorly understood. Many reactions mechanisms have been proposed on chemical grounds, but there is insufficient biochemical evidence to permit a choice among these proposals. A quantity of negative evidence has accumulated suggesting that group X migration does not involve alkylation of the cobalt of cobalamin by the substrate, but in the absence of firm data supporting an alternative mechanism, even this weak conclusion must be regarded as provisional.

The Role of Oxygen Radicals in Microbial Killing by Phagocytes

Recognition of the possibility that phagocytes might employ oxygen radicals as microbicidal agents dates from the discovery by McCord and Fridovich (1969) of the superoxide dismutases, a nearly ubiquitous group of enzymes, which catalyze the destruction of the oxygen radical O2 — (superoxide) according to the reaction:

Chromatin structure during bleomycin-induced DNA damage and repair

2{O_{{{{\dot{2}}^{ - }}}}} + 2H{}^{ + } \to {O_{2}} + {H_{2}}{O_{2}}