Reactive oxygen species in the killing of Pseudomonas aeruginosa by human leukocytes

Pseudomonas aeruginosa (PA) infects hosts with compromised host defenses. An important defense mechanism is the generation of reactive oxygen species (ROS) by white blood cells (WBCs). What roles do ROS play in host defense against PA? Human WBCs killed PA in vitro, and they generated a respiratory burst as measured by the production of H₂O₂. ROS efficiently killed PA; in acellular assays, less than 10mm of H₂O₂ or OCl^- eliminated all bacteria in 90 min. However, WBCs with suppressed production of ROS (caused by hypoxia) killed PA normally. In addition, none of the antioxidants vitamin C, N-acetylcysteine, superoxide dismutase, or catalase affected PA killing by WBCs. Thus, PA stimulates WBCs to produce ROS, which can kill the bacteria, but disturbances of WBC ROS production do not interfere with the killing of PA. WBCs have robust, redundant mechanisms for PA elimination.     

Effects of granulocytes on human neuroblastoma cells measured by chemiluminescence and chromium-51 release assay

We investigated whether polymorphonuclear leukocytes (PMN) are able to kill human neuroblastoma cells either directly or if coated with antibody MAb 14.18 that recognizes ganglioside GD₂ present on the cell surface of most neuroblastoma cells. Neuroblastoma cells could not be destroyed directly, whereas in the antibody-dependent reaction (ADCC-reaction) they were easily eliminated. In order to answer the question whether reactive oxygen intermediates are involved in this process, chemiluminescence measurements were performed. Compared to the signals that could be measured using opsonized zymosan as stimulus, only weak CL-signals could be registered during the ADCC reaction. Pretreatment of PMN with granulocyte-macrophage colony stimulating factor (GM-CSF) enhanced the CL-signals, catalase and SOD reduced it; however, cell killing was only slightly influenced in the presence of catalase and superoxide dismutase. These data suggested that reactive oxygen compounds do not play a prominent role in the killing process. Definitive evidence for this suggestion could be obtained using PMN from a patient with chronic granulomatous disease (CGD): MAb 14.18 coated neuroblastoma cells could be killed effectively, but no CL-signal could be registered, either in the ADCC-reaction or using opsonized zymosan as stimulus.     

Oxidative cytochemistry in phagocytosis: the interface between structure and function

Summary The PMN is exquisitely designed to combat invading micro-organisms. The relationship between structure and function is nowhere more evident than in this cell type. The elaborate biochemical machinery which the PMNs possess for killing ingested micro-organisms works as a highly integrated system, with each step occurring in sequence and at a particular site.In the past decade or so it has become apparent that the Klebanoff system (myeloperoxidase-halide-H₂O₂) and possibly other active O₂ species as well, play an important role in the bactericidal activity of PMNs. Application of cytochemical techniques for oxidative enzymes and for end-products of oxidative reactions has localized the sites within the phagocytosing or stimulated PMN at which these various components of the cidal systems are active and generated. In this fashion, biochemical data have been not only confirmed, but in several instances, the cytochemical approach has led the way in extending our knowledge and thinking regarding PMN metabolism and cidal functions.In our laboratory we have studied the bactericidal machinery of PMNs by cytochemical means. We have established, at the ultrastructural level, that the myeloperoxidase-containing azurophil granules fuse with the phagosome membrane and empty their contents into the phagosome (Baehneret al., 1969). We have shown that H₂O₂ is generated within the phagosome (Briggset al., 1975b). This established that the myeloperoxidase-H₂O₂ system could work within the phagosome, since both of these components are present following phagocytosis. We determined that H₂O₂ could be detected on the cell surface and within the phagosome following phagocytic stimulation of NADH oxidase activity (Briggset al., 1975a). The cell surface localization of H₂O₂ was an important finding since the phagosome membrane is derived from the plasmalemma. Thus internalization of the plasmalemma, with components capable of generating H₂O₂, can explain the presence of H₂O₂ within the phagosome. We have also shown that when PMNs are treated with non-particulate stimuli of the respiratory burst, similar results are found, that is, H₂O₂ is present on the cell surface and within vesicles, which are presumed to be of surface origin (Badweyet al., 1980). We have shown that D-amino acid oxidase, another enzyme capable of generating H₂O₂ is cytochemically demonstrable and that it can utilize cell wall components of ingested bacteria as substrates for enzyme activity (Robinsonet al., 1978).The PMNs from CGD patients do not kill certain bacteria. This inability to kill bacteria is related to the low levels of H₂O₂ produced during phagocytosis. Using the cerium reactioon we determined that PMN from CGD patients produce little cytochemically detectable H₂O₂ and that what little is present is restricted to the phagosome (Briggset al., 1977).Some PMNs contain other oxidases which are capable of generating H₂O₂ and O ₂ ^– from O₂ consumed during phagocytosis. The guinea-pig PMN (but not human) has an unusual aldehyde oxidase. Cytochemically the aldehyde oxidase activity is restricted to the phagosome (Robinsonet al., 1979).We have also developed a method for localization of sites of O ₂ ^– production following stimulation. In phorbol myristate acetate-stimulated PMNs, reaction product for O ₂ ^– is present within surface-derived vesicles, and in some cases, on the cell surface.Cytochemical detection of enzymes and products of enzymatic activity (H₂O₂ and O ₂ ^– ) associated with stimulation of the respiratory burst in PMN has thus provided further evidence for the importance of active oxygen species in phagocytosis. Furthermore, the site-specific information obtained from cytochemistry has provided an important link in understanding the structure-function interplay associated with phagocytosis in PMNs.It should be realized, however, that the cytochemical methods we have utilized detect in most instances the end product of an enzymatic reaction (for example, H₂O₂) and not the site of the enzyme itself. This is important, for instance in the case of H₂O₂, because this entity appears to begenerated on the surface of the plasmalemma or on the luminal surface of the phagosomal membrane. However, the enzyme responsible may well be situated on the cytoplasmic side of these membranes, and the generation of the H₂O₂ may involve an electron shuttle across the membrane. Such a mechanism may involve cytochrome and quinone compounds as carriers (Segal & Jones, 1979; Millardet al., 1979). Experiments are now being designed to localize the sites of the enzymesper se by immunocytochemistry. This approach should help resolve these important questions.The 1980Histochemical Journal Lecture at the invitation of the Histochemistry and Cytochemistry Section of the Royal Microscopical Society given by Dr M. J. Karnovsky to a Symposium on Cell Uptake and Transport held at the Sixth International Histochemistry and Cytochemistry Congress in Brighton, England, on 21 August, 1980.     

Exposure of Phytopathogenic Xanthomonas spp. to Lethal Concentrations of Multiple Oxidants Affects Bacterial Survival in a Complex Manner

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1,000-fold; conversely, treatment of cells with menadione plus H₂O₂ resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H₂O₂ and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H₂O₂ and tert-butyl hydroperoxide plus H₂O₂. These data suggest that H₂O₂ is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H₂O₂-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.     

Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.     

Enzyme immunosensor. III. Amperometric determination of human chorionic gonadotropin by membrane-bound antibody.

An enzyme immunosensor was constructed for the determination of human chorionic gonadotropin (HCG), which is a hormone and an important diagnostic measure of pregnancy. An antibody to HCG was immobilized to a membrane. The antibody-bound membrane was placed onto an oxygen probe so as to react with HCG either specifically or selectively. Catalase, which catalyzes the decomposition of H₂O₂ into H₂O and O₂, was used to label HCG. Nonlabeled HCG to be assayed and catalase-labeled HCG were competitively reacted with the membrane-bound antibody of the sensor to form an antigen-antibody complex on the membrane surface. After the removal of nonspecifically adsorbed HCG, the sensor was contacted with a H₂O₂ solution. The membrane-adsorbed catalase enzymatically generated oxygen with a resulting increase in cathodic current of the sensor. The HCG concentration was determined from the initial rate of the current increase. The enzyme immunosensor was applied to the determination of HCG in the concentration range of 2 × 10^?2 to 10² IU/ml.     

Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H₂O₂ and superoxide (O₂⁻) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H₂O₂ and O₂⁻ production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 × 10⁸ colony-forming units/mL of incompatible Pseudomonas corrugata or 158 μm HgCl₂. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 μm HgCl₂ did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O₂⁻ to H₂O₂, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P ≥ 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H₂O₂ is the substrate for the luminol reaction. Addition of live bacteria or HgCl₂ (25 and 158 μm) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 μm HgCl₂ did not develop an HR. These results suggest that an oxidative burst consisting of H₂O₂ and O₂⁻ does occur during these two plant defense responses, but it may not be a necessary element of the signaling system for HR and phytoalexin formation.     

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Cytochemistry and reactive oxygen species: a retrospective

This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O^·– ₂, H₂O₂, and the H₂O₂-myeloperoxidase (MPO)-halide system. The H₂O₂-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H₂O₂, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H₂O₂ to yield oxidized DAB, H₂O₂ was localized to phagolysosomes. The subcellular sites of H₂O₂ generation were shown using cerium ions which react with H₂O₂ and precipitate electron opaque cerium perhydroxides (Ce(OH)₂OOH and Ce(OH)₃OOH). The results suggested that NADPH oxidase is associated with the plasmalemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H₂O₂ in the phagosome. As O^·– ₂ is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O^·– ₂ oxidizes Mn²⁺ to Mn³⁺, and Mn³⁺ oxidizes DAB, a medium containing DAB-Mn²⁺ was used to localize sites of O^·– ₂ production in stimulated PMN. The localizations were, as expected, similar to those for H₂O₂. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.Presented at the 36th Symposium of the Society for Histochemistry, 22 September 1994, Heidelberg, Germany     

Overlapping and Complementary Oxidative Stress Defense Mechanisms in Nontypeable Haemophilus influenzae

The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H₂O₂), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX, and dps. The hktE mutant and a pgdX hktE double mutant were more sensitive than the parent to killing by H₂O₂. Conversely, the pgdX mutant was more resistant to H₂O₂ due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H₂O₂-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.     

Peroxynitrite and hydrogen peroxide elicit similar cellular stress responses mediated by the Ccp1 sensor protein

Peroxynitrite [ONOO(H)] is an oxidant associated with deleterious effects in cells. Because it is an inorganic peroxide that reacts rapidly with peroxidases, we speculated that cells may respond to ONOO(H) and H₂O₂ challenge in a similar manner. We exposed yeast cells to SIN-1, a well-characterized ONOO(H) generator, and observed stimulation of catalase and peroxiredoxin (Prx) activities. Previously, we reported that H₂O₂ challenge increases these activities in wild-type cells and in cells producing the hyperactive mutant H₂O₂ sensor Ccp1^W191F but not in Ccp1-knockout cells (ccp1Δ). We find here that the response of ccp1Δ and ccp1^W191F cells to SIN-1 mirrors that to H₂O₂, identifying Ccp1 as a sensor of both peroxides. SIN-1 simultaneously releases ^•NO and O₂^•−, which react to form ONOO(H), but exposure of the three strains separately to an ^•NO donor (spermine-NONOate) or an O₂^•− generator (paraquat) mainly depresses catalase or Prx activity, whereas co-challenge with the NONOate and paraquat stimulates these activities. Because Ccp1 appears to sense ONOO(H) in cells, we examined its reaction with ONOO(H) in vitro and found that peroxynitrous acid (ONOOH) rapidly (k₂>10⁶ M⁻¹ s⁻¹) oxidizes purified Ccp1 to an intermediate with spectral and ferrocytochrome-oxidizing properties indistinguishable from those of its well-characterized compound I formed with H₂O₂. Importantly, the nitrite released from ONOOH is not oxidized to ^•NO₂ by Ccp1^׳s compound I, unlike peroxidases involved in immune defense. Overall, our results reveal that yeast cells mount a common antioxidant response to ONOO(H) and H₂O₂, with Ccp1 playing a pivotal role as an inorganic peroxide sensor.     

Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to ¹²⁵I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10^?8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-μm-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial ¹²⁵I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial ¹²⁵I-albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H₂O₂), did not alter the transendothelial ¹²⁵I-albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H₂O₂ generated during PMN activation, did not alter the increase in transendothelial ¹²⁵I-clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial ¹²⁵I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H₂O₂, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants. © 1993 Wiley-Liss, Inc.     

The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

Enhanced killing of penicillin-treated gram-positive cocci by human granulocytes: role of bacterial autolysins, catalase, and granulocyte oxidative pathways

Staphylococci pretreated with subminimal inhibitory concentrations (subMIC) of cell-wall active antibiotics exhibit increased susceptibility to killing by human polymorphonuclear leukocytes (PMNs), even when phagosome information is impaired by the mold metabolite, cytochalasin B. To investigate the role of specific bacterial factors in the process, studies were carried out with organisms lacking catalase (streptococci) or cell-wall autolytic enzymes and compared to findings with Staphylococcus aureus 502A. Neutrophil factors were studied using inhibitors, oxygen radical scavengers, myeloperoxidase (MPO)-deficient PMNs, or PMNs from a patient with chronic granulomatous disease (CGD). Documentation of the enhanced susceptibility of the streptococcal strains to killing by PMNs following subMIC penicillin pretreatment required the use of cytochalasin B. Enhancement of killing occurred independent of the presence or absence of bacterial autolysins or catalase. SubMIC penicillin pretreatment of S. pneumoniae R36A specifically promoted the susceptibility of these organisms to killing by myeloperoxidase (MPO)-mediated mechanisms (enhancement lost using MPO-deficient or azide-treated cells). Factors other than MPO or toxic oxygen products generated by the PMN respiratory burst are responsible for enhanced killing of penicillin-pretreated S. aureus 502A (enhancement preserved using MPO-deficient, azide-treated, or chronic granulomatous disease patient cells). These studies define methods to study the interaction of antimicrobial agents and PMNs in the killing of microorganisms. They also demonstrate that penicillin treatment can change the susceptibility of gram-positive cocci to the action of specific PMN microbicidal mechanisms. The mechanism of the enhancement appears to be bacterial strain-dependent and not predictable by bacterial autolysin or catalase activity.     

兼性厌氧细菌硫化氢的产生机理及其生理功能

硫化氢(H_2S)是继一氧化氮(NO)和一氧化碳(CO)后发现的第3种气态信号分子,但其细菌生理学研究才刚刚起步。本文根据作者对奥内达希瓦氏菌的研究,结合新近文献,就细菌的H_2S产生机理及其生理功能作了较为全面的阐述。细菌的H_2S产生途径主要有2条,一是通过降解半胱氨酸产生,二是通过厌氧呼吸产生。产生的H_2S除可为互生性微生物提供能源、供氢体和无机矿质营养外,还具有抑制竞争性微生物的生长,有效占领生态位的作用。H_2S在氧化应答中也起着重要的作用,一方面可抑制过氧化氢酶活性,增加过氧化氢对细菌的杀灭效果;另一方面可作为信号分子激活细菌的氧化应答,诱导拮抗系统的表达,保护细胞免受氧化损伤。这两种看似"矛盾"的作用与H_2S的处理时间有关:短时间处理以抑制为主,而延长处理时间则以保护为主。细菌H_2S产生机理及生理功能的阐明可为硫元素生物地球化学循环规律的揭示和感染性病原细菌的控制提供有益的参考。     

DNA strand scission by enzymically generated oxygen radicals

Col E1 DNA suffers strand scission when exposed to xanthine oxidase acting aerobically on xanthine. Strand scission was prevented by low levels of superoxide dismutase or of catalase. Mannitol, benzoate, or histidine, which scavenge OH · but which react with neither O₂^? nor H₂O₂, also prevented strand scission. Replacement of 0.1 mm ethylenediaminetetraacetate by 0.1 mm diethylenetriaminepentaacetate prevented strand scission. Three mechanisms for the production of OH ·, or of a comparably powerful oxidant, by metal-catalyzed interaction of O₂^? with H₂O₂, are proposed.     

Effects of α-ketoglutarate on neutrophil intracellular amino and α-keto acid profiles and ROS production

The aim of this study was to determine the effects of α-ketoglutarate on neutrophil (PMN), free α-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O₂ ^?), hydrogen peroxide (H₂O₂)] and released myeloperoxidase (MPO) acitivity. Exogenous α-ketoglutarate significantly increased PMN α-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular α-ketoglutarate changes, increases in O₂ ^– formation, H₂O₂-generation and MPO acitivity have also been observed. We therefore believe that α-ketoglutarate is important for affecting PMN “susceptible free amino- and α-keto acid pools” although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic α-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.     

Catalase has only a minor role in protection against near-ultraviolet radiation damage in bacteria

Summary In bacterial cells near-ultraviolet radiation (NUV) generates H₂O₂ which can be decomposed by endogenous catalase to H₂O and O₂. To assess the roles of H₂O₂ and catalase in NUV lethality, we manipulated the amount of intracellular catalase (a) by the use of mutant and plasmid strains with altered endogenous catalase, (b) physiologically, by the addition of glucose, and (c) by induction of catalase synthesis with oxidizing agents. Not only was there no direct correlation between NUV-resistance and catalase activity, but in some cases the correlation was inverse. Also, while there was correlation between NUV and H₂O₂ sensitivity for most strains tested, there were a number of exceptions which indicates that the modes of killing were different for the two agents.     

Bactericidal Activity of Catecholamine Copper Complexes

Washed or growing E. coli cells are killed by epinephrine, norepinephrine or dopamine in the presence of non lethal concentrations of Cu(II). Killing is enhanced by anoxia and by sublethal Concentrations of H₂O₁. The rate of killing is proportional to the rate of catecholamine oxidation. The copper epinephrine complex binds to E. coli cells, induces membrane damage and depletion of the cellular ATP pool. The cells may be partially protected by SOD or catalase but not by OH radical scavengers. Addition of H²O₂ to cells which were sensitized by preincubation with the epinephrine-copper complex, causes rapid killing and DNA degradation. Sensitized cells are not protected by BSA.