Showing papers by "Myron S. Cohen published in 1989"

Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells.

Abstract: During phagocytosis, phagocytic cells generate superoxide and other reactive oxygen species, which are involved in antibacterial activity. However, many bacteria possess antioxidant defenses that may explain their survival in inflammatory foci. These defenses include antioxidant enzymes such as superoxide dismutase and catalase, DNA repair systems, scavenging substrates, and competition with phagocytes for molecular oxygen. These defenses are probably coordinated, and different responses occur with different reactive oxygen species. Escherichia coli and Salmonella typhimurium mutants have allowed the demonstration of a variety of critical genes for enzymatic defense and DNA repair, as well as an oxyR regulon system. In more complex systems, the conditions found in inflammatory foci, such as decreasing glucose and the production of lactate, enhance bacterial catalase production and resistance to hydrogen peroxide. Resistance and adaptation to phagocyte-derived oxidant stress are critical aspects of bacterial pathogenesis.

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity

Abstract: Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 μM and is maximal at 1 μM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.

Neutrophil degranulation inhibits potential hydroxyl-radical formation. Relative impact of myeloperoxidase and lactoferrin release on hydroxyl-radical production by iron-supplemented neutrophils assessed by spin-trapping techniques

Abstract: Hydroxyl radical (.OH) formation by neutrophils in vitro requires exogenous iron. Two recent studies [Britigan, Rosen, Thompson, Chai & Cohen (1986) J. Biol. Chem. 261, 17026-17032; Winterbourn (1987) J. Clin. Invest. 78, 545-550] both reported that neutrophil degranulation could potentially inhibit the formation of .OH, but differed in their conclusions as to the responsible factor, myeloperoxidase (MPO) or lactoferrin (LF). By using a previously developed spin-trapping system which allows specific on-line detection of superoxide anion (O2-) and .OH production, the impact of MPO and LF release on neutrophil .OH production was compared. When iron-diethylenetriaminepenta-acetic acid-supplemented neutrophils were stimulated with phorbol myristate acetate or opsonized zymosan, .OH formation occurred, but terminated prematurely in spite of continued O2- generation. Inhibition of MPO by azide increased the magnitude, but not the duration, of .OH formation. No azide effect was noted when MPO-deficient neutrophils were used. Anti-LF antibody increased both the magnitude and duration of .OH generation. Pretreatment of neutrophils with cytochalasin B to prevent phagosome formation did not alter the relative impact of azide or anti-LF on neutrophil .OH production. An effect of azide or anti-LF on spin-trapped-adduct stability was eliminated as a confounding factor. These data indicate that neutrophils possess two mechanisms for limiting .OH production. Implications for neutrophil-derived oxidant damage are discussed.

Oxidant stress in Neisseria gonorrhoeae: adaptation and effects on L-(+)-lactate dehydrogenase activity.

Abstract: Neisseria gonorrhoeae, an obligate human pathogen, is subjected to oxidant stress when attacked by O2 reduction products formed by neutrophils. In this study, exposure of gonococci to sublethal concentrations of superoxide and hydrogen peroxide (and related O-centered radicals) resulted in phenotypic resistance to oxidant stress. Adaptation required new protein formation but was not related to increases in superoxide dismutase or catalase. We have previously demonstrated that gonococci use phagocyte-derived L-(+)-lactate. Oxidant stress of greater magnitude than that required for adaptation led to a generalized increase in bacterial metabolism, particularly in L-(+)- and D-(-)-lactate utilization and lactate dehydrogenase activity. Increased lactate utilization required new protein synthesis. These results suggest the possibility that lactate metabolism is of importance to N. gonorrhoeae subjected to oxidant stress. Use of lct mutant organisms unable to use L-(+)-lactate should allow examination of this hypothesis.

Detection of free radicals as a consequence of dog tracheal epithelial cellular xenobiotic metabolism.

Abstract: 1. Spin-trapping techniques have been used to examine the metabolism of three xenobiotics known to produce free radicals during their metabolism. Reaction with oxygen generated superoxide, the location of which was dependent upon the xenobiotic.2. Paraquat was metabolized by dog trachea epithelial cells under anaerobiosis to the paraquat free radical, some of which diffused into the extracellular milieu. With the addition of oxygen, superoxide was spin-trapped both intra- and extracellularly.3. When menadione was metabolized by epithelial cells, superoxide was spin-trapped within the cell and in the surrounding media. However, in this case, extracellular superoxide arose as the result of the disproportionation reaction of menadione and menadiol, resulting from DT-diaphorase reduction of menadione followed by diffusion into extracellular space, to give the menadione semiquinone. Reduction of oxygen resulted in formation of superoxide.4. For nitrazepam, only intracellular superoxide was generated, resulting...

The role of hydroxyl radical in chromosomal and plasmid damage in Neisseria gonorrhoeae in vivo.

Abstract: Viable Neisseria gonorrhoeae exposed to streptonigrin generate intracellular hydroxyl radical detected by spin-trapping with 5,5-dimethyl-l-pyrroline-N-oxide; gonococci exposed to paraquat generate primarily superoxide (J Biol Chem, 262: 13404-143048, 1987) The use of streptonigrin and paraquat provide a model with which to examine the action and site(s) of hydroxyl radical-mediated damage N gonorrhoeae exposed to streptonigrin, but not paraquat, developed extensive chromosomal, plasmid, and RNA damage Addition of excess Fe+3 to the reaction mixture enhanced intracellular hydroxyl radical formation by paraquat, detectable as DNA damage Desferal and dimethyl sulfoxide allowed approximately 25% of protection of plasmid DNA damage as judged by linear scanning densitometry These results demonstrate DNA and RNA damage in viable organisms exposed to intracellular redox stress and confirm the critical role of hydroxyl radical in this process

Prospective comparative trial of short course (four day) and continuous tobramycin in combination with cefoperazone or mezlocillin in febrile, granulocytopenic patients

Abstract: In a prospective, randomized trial of 195 febrile episodes in granulocytopenic patients short course aminoglycoside treatment (initial tobramycin and cefoperazone followed by tobramycin discontinuation at day four of therapy) was compared with two regimens (tobramycin plus cefoperazone and tobramycin plus mezlocillin) in which both drugs were continued for up to 26 days. All regimens were successful as empirical therapy with comparable response rates of just over seventy per cent. Fifty-three per cent of the initial episodes of fever were related to documented infections which responded less well (P = 0.007) than unexplained fever. Patients with bacteraemia, pneumonia or Gram-positive aerobic or Pseudomonas aeruginosa infections responded poorly to all regimens. The recovery from granulocytopenia was the most important determinant of successful response. Aminoglycoside discontinuation followed by cefoperazone monotherapy after day four was statistically as effective as the combination regimens. Short course tobramycin therapy eliminated the nephrotoxicity seen in the combination limbs. The use of cefoperazone was not associated with an increased incidence of hypoprothrombinemia; however, the only three bleeding episodes occurred in patients given cefoperazone but not vitamin K. Short course aminoglycoside therapy will reduce cost and nephrotoxicity when compared with prolonged combination therapy and should be further explored in this setting, with use of different agents and comparison with monotherapy.

Energy is required for maximal adherence of Neisseria gonorrhoeae to phagocytic and nonphagocytic cells.

Abstract: The possibility that gonococcal energy might play a role in the interaction of Neisseria gonorrhoeae with both phagocytic and nonphagocytic cells was examined. Respiratory chain inhibitors including KCN and amobarbital resulted in reduction in gonococcal association with human neutrophils. Similar results were seen with HeLa cells and the human promyelocytic (HL-60) cell line. Identical conditions did not affect the opsonin-dependent association of Staphylococcus aureus with the same cell types. New protein synthesis by gonococci did not account for the observed reduction in association. These results suggest that energy is needed for maximal opsonin-independent association of gonococci with mammalian cells.