Chitooligosaccharides Protect Human Embryonic Hepatocytes Against Oxidative Stress Induced by Hydrogen Peroxide

Chitooligosaccharides (COS) has many biological activities, such as antitumor activity and hepatoprotective effect. Herein, we investigated the protective effect of COS against hydrogen peroxide (H₂O₂)-induced oxidative stress on human embryonic hepatocytes (L02 cells) and its scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl radical in vitro. The results showed that the lost cell viability induced by H₂O₂ was markedly restored after 24 h pre-incubation with COS (0.1–0.4 mg/ml). This rescue effect could be related to the antioxidant property of COS, in which we showed that the radical scavenging activity of COS reached 80% at concentration of 2 mg/ml. In addition, COS could prevent cell apoptosis induced by H₂O₂, as shown by the inhibition of the cleavage of poly (adenosine diphosphate-ribose) polymerase and increased expression of the anti-apoptotic protein Bcl-xL. Furthermore, we have utilized confocal laser microscopy to observe cellular uptake of COS, an important step for COS to exert its effects on target cells. Taken together, our findings suggested that COS could effectively protect L02 cells against oxidative stress, which might be useful in clinical setting during the treatment of oxidative stress-related liver damages.     

The role of MAC1 in diesel exhaust particle‐induced microglial activation and loss of dopaminergic neuron function

Increasing reports support that air pollution causes neuroinflammation and is linked to central nervous system (CNS) disease/damage. Diesel exhaust particles (DEP) are a major component of urban air pollution, which has been linked to microglial activation and Parkinson's disease‐like pathology. To begin to address how DEP may exert CNS effects, microglia and neuron‐glia cultures were treated with either nanometer‐sized DEP (< 0.22 μM; 50 μg/mL), ultrafine carbon black (ufCB, 50 μg/mL), or DEP extracts (eDEP; from 50 μg/mL DEP), and the effect of microglial activation and dopaminergic (DA) neuron function was assessed. All three treatments showed enhanced ameboid microglia morphology, increased H₂O₂ production, and decreased DA uptake. Mechanistic inquiry revealed that the scavenger receptor inhibitor fucoidan blocked DEP internalization in microglia, but failed to alter DEP‐induced H₂O₂ production in microglia. However, pre‐treatment with the MAC1/CD11b inhibitor antibody blocked microglial H₂O₂ production in response to DEP. MAC1^?/? mesencephalic neuron‐glia cultures were protected from DEP‐induced loss of DA neuron function, as measured by DA uptake. These findings support that DEP may activate microglia through multiple mechanisms, where scavenger receptors regulate internalization of DEP and the MAC1 receptor is mandatory for both DEP‐induced microglial H₂O₂ production and loss of DA neuron function.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Activation of ascorbate-glutathione cycle inArabidopsis leaves in response to aminotriazole

Aminotriazole(AT)-induced changes in growth, hydrogen peroxide content and activities of H₂O₂-scavenging antioxidant enzymes were investigated in the growing leaves ofArabidopsis plants (Arabidopsis thaliana cv Columbia). Catalase activity of rosette leaves was reduced by 65% with an application of 0.1 mM AT (a herbicide known as a catalase inhibitor), whereas the leaf growth and H₂O₂ content were almost unaffected. However, an approximate 1.6 to 2-fold increase in cytosolic ascorbate peroxidase (APX) activity concomitant with a substantial activation of glutathione reductase (GR) (approx. 22% increase) was observed during leaf growth in the presence of 0.1 mM AT. The activity of cytosolic APX in leaves was also increased by 1.8-fold with an application of exogenous 2 mM paraquat (an inducer of H₂O₂ production in plant cells) in the absence of AT. These results collectively suggest that (a) cytosolic APX and GR operate to activate an ascorbate-glutathione cycle for the removal of H₂O₂ under severe catalase deactivation, and (b) the expression of APX seems to be regulated by a change of the endogenous H₂O₂ level in leaf cells.     

INJECTION OF 6-HYDROXYDOPAMINE AND HYDROGEN PEROXIDE INTO THE SUBSTANTIA NIGRA AND LATERAL VENTRICLE OF THE CAT: SPECIFIC AND NONSPECIFIC EFFECTS ON STRIATAL BIOGENIC AMINES1

Attempting to clarify the mechanism by which intracerabral injection of 6-hydroxydopamine (60HDA) reduces catecholamines in the caudate nucleus (CN), we have tested two hypotheses: (1) 60HDA specifically attacks catecholaminergic neurons; (2) 60HDA liberates hydrogen peroxide (H₂O₂) which destroys neurons indiscriminately. To this end, we have injected high or low doses of 60HDA or equimolar amounts of H₂O₂ stereotaxically into the substantia nigra (SN) or the lateral ventricle of cats and have placed electrocoagulative lesions in the SN. We determined the CN levels of dopamine (DA), norepinephrine (NE) and serotonin (5HT) 7-10 days later. Nigral injections of high doses (8 μ mol) of either agent or low doses (80 nmol) of 60HDA decreased both DA and NE and induced similar histologic damage in the SN with neuronal drop-out at the periphery of the lesions. Injection of 80 nmol of H₂O₂ into the SN did not decrease CN amine levels and did not produce histologic damage in the SN. Electrocoagulation of the SN decreased CN DA and NE, but the histologic lesions failed to show any peripheral neuronal drop-out. Ventricular injections of high doses (16 μmol) of 60HDA or H₂O₂ reduced not only DA and NE but also 5HT levels in the ipsilateral CN. Low intraventricular doses (0-16 μmol) of 60HDA decreased only DA and NE without affecting 5HT levels in the CN whereas 0.16 μmol of H₂O₂ had no effect on any of the CN amines. The catecholamine-depleting effects of low doses (80 nmol) of 60HDA were significantly potentiated by inhibiting brain monoamine oxidase by 90 percent or more at the time and site of injection of 60HDA. These results suggest that the extracellular liberation of H₂O₂ from 60HDA could explain some possibly nonspecific effects of high doses of 60HDA; at lower doses, however, 60HDA may act via selective uptake into catecholaminergic neurons with subsequent intracellular release of H₂O₂.     

Enhanced Killing of Acanthamoeba Cysts with a Plant Peroxidase-Hydrogen Peroxide-Halide Antimicrobial System

The activity of H₂O₂ against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H₂O₂, and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H₂O₂, enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H₂O₂ was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H₂O₂ was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H₂O₂ in contact lens care systems, the enhanced 2% H₂O₂ system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H₂O₂ occurred by 4 h. In contrast, 2% H₂O₂ alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H₂O₂ contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis.     

Effect of Hydrogen Peroxide on Immersion Challenge of Rainbow Trout Fry with Flavobacterium psychrophilum

An experimental model for immersion challenge of rainbow trout fry (Oncorhynchus mykiss) with Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome and bacterial cold water disease was established in the present study. Although injection-based infection models are reliable and produce high levels of mortality attempts to establish a reproducible immersion model have been less successful. Various concentrations of hydrogen peroxide (H₂O₂) were evaluated before being used as a pre-treatment stressor prior to immersion exposure to F. psychrophilum. H₂O₂ accelerated the onset of mortality and increased mortality approximately two-fold; from 9.1% to 19.2% and from 14.7% to 30.3% in two separate experiments. Clinical signs observed in the infected fish corresponded to symptoms characteristically seen during natural outbreaks. These findings indicate that pre-treatment with H₂O₂ can increase the level of mortality in rainbow trout fry after exposure to F. psychrophilum.     

17β-Estradiol and steady-state concentrations of H2O2: antiapoptotic effect in endometrial cells from patients with endometriosis

Increased levels of hydrogen peroxide (H₂O₂) can initiate protective responses to limit or repair oxidative damage. However, H₂O₂ signals also fine-tune responses to growth factors and cytokines controlling cell division, differentiation, and proliferation. Because 17β-estradiol (E₂) also plays important roles in these processes, and is considered a major risk factor in the development and progression of endometriosis, this study evaluated whether E₂ has an antiapoptotic effect on oxidative stress in endometrial cells in combination with steady-state H₂O₂ levels ([H₂O₂]ss). Endometrial stromal cells were prepared from the eutopic endometrium of 18 women with and without endometriosis to produce primary cells. These cells were stimulated with E₂ for 20 h, exposed to [H₂O₂]ss, and examined for cell viability, proliferation, and apoptosis. The endometrial cells from women with endometriosis maintained the steady state for 120 min at high H₂O₂ concentrations. When they were pretreated with E₂ and exposed to [H₂O₂]ss, a decrease in apoptosis level was observed compared to the control cells (p<0.01). The endometrial cells from patients with endometriosis subjected to both E₂ and [H₂O₂]ss showed increased ERK phosphorylation. These findings suggested that H₂O₂ is a signaling molecule that downregulates apoptosis in endometrial cells, supporting the fact that endometriosis, albeit a benign disease, shares some features with cancer such as decreased catalase levels. These results link the E₂ effects on [H₂O₂]ss to resistance to apoptosis and progression of endometriosis.     

The efficacy and sterilisation of human decellularised dermal allografts with combinations of cupric ions and hydrogen peroxide

Decellularised tissue allografts have been used in reconstructive surgical applications and transplantation for many years. Some of the current methods of sterilisation have a detrimental effect on the tissue graft structure and function. The anti-microbial activity of cupric ions and hydrogen peroxide (H₂O₂) are well known however their combined application is not currently utilised as a decontamination agent in the tissue banking world sector. The aim of this study was to determine the combined concentrations of copper chloride (CuCl₂) and H₂O₂ that have the optimal bactericidal and sporicidal activity on decellularised (dCELL) human dermis. The first part of this study established the decimal reduction time (D-value) of CuCl₂ (0.1 mg/L and 1 mg/L) together with H₂O₂ (0.01, 0.1, 0.5 and 1%) for Staphylococcus epidermidis, Escherichia coli and Bacillus subtilis spores. The second part of this study identified the most effective CuCl₂ and H₂O₂ concentration that decontaminated dCELL human dermis inoculated with these pathogens. Of all the concentrations tested, 0.1 mg/L CuCl₂ in combination with 1% H₂O₂ had the shortest D-value; S. epidermidis D = 3.15 min, E. coli D = 2.62 min and B. subtilis spores D = 18.05 min. However when adsorbed onto dCELL dermis, S. epidermidis and E. coli were more susceptible to 1 mg/L CuCl₂ together with 0.5% H₂O₂. These studies show promise of CuCl₂–H₂O₂ formulations as potential sterilants for decellularised dermal allografts.     

Use of Carbon Oxysulfide, a Structural Analog of CO(2), to Study Active CO(2) Transport in the Cyanobacterium Synechococcus UTEX 625

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO₂. Although hydrolysis of COS (to CO₂ and H₂S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO₂ transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO₂ and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H₂S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H₂S. The active transport of CO₂ was inhibited by COS in an apparently competitive manner. When Na⁺-dependent HCO₃⁻ transport was allowed in the presence of COS, the extracellular [CO₂] rose considerably above the equilibrium level. This CO₂ appearing in the medium was derived from the dehydration of transported HCO₃⁻ and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO₂ was inhibited. These results showed that the Na⁺-dependent HCO₃⁻ transport was not inhibited by COS, whereas active CO₂ transport was inhibited. When COS was removed by gassing with N₂, a normal pattern of CO₂ uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO₃⁻ transport or CO₂ fixation. The steady state rate of CO₂ fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO₂ from the cells that occurred when CO₂ uptake was inhibited by COS. Neither CS₂ nor N₂O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO₂ transport.     

A dissection of the effects of ethylene,H2O2 and high irradiance on antioxidants and several genes associated with stress and senescence in tobacco leaves

Ethylene and hydrogen peroxide are involved in the modulation of stress responses in plants, but their interrelation is not well understood. This work was designed to find differences between the actions of ethylene and H₂O₂ on antioxidants and senescence markers. Leaves of Nicotiana tabacum were sprayed with H₂O₂ or with ethephon (precursor of ethylene). To find the possible modulation of responses to acute abiotic stress, ethephon- and H₂O₂-sprayed leaves were further subjected to high irradiance (HL). The application of H₂O₂ strongly stimulated ethylene synthesis (ACC). Ethylene and H₂O₂, as single factors, stimulated the trolox equivalent antioxidant capacity (TEAC) and the activity of catalase (CAT), in contrast to HL alone (stimulation of nonspecific peroxidases and the total glutathione pool). However, after combined treatments (ethylene + HL and H₂O₂ + HL), the stimulatory action of H₂O₂ was related to TEAC and CAT activity, while the application of ethylene stimulated the total glutathione pool. Hydrogen peroxide enhanced the expression of the three CAT genes (Cat1, Cat2 and Cat3), in contrast to ethylene (Cat2 and Cat3) and HL (Cat1). In regard to the markers of senescence and pathogenesis the most pronounced difference between the actions of ethylene and H₂O₂, as single factors, was related to NPR1, whereas when leaf spraying was combined with HL, differences were found at WRKY53 and PR1a. HL reversed the stimulatory effects of H₂O₂/ethylene-driven enhancements of the expression of several genes (Cat1, Cat2, NPR1, WRKY53). These results show that multiple stressors, as usually encountered by plants in nature, may largely change those expression patterns of genes determined in a single factor analysis. Moreover, the actions of HL (often considered the internal H₂O₂ trigger) and of exogenous H₂O₂ on gene expression are clearly different.     

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b₅₆₂ by 71%, in an [O₂] independent manner. Hyperbolic increases in O₂^•- (up to 1.3±0.1 nmol/min. mg protein) and H₂O₂ (up to 0.64±0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O₂^•-/H₂O₂ ratio was 1.98 in accordance with the stoichiometry of the O₂^•- disproportionation. Moreover, H₂O₂ production was increased by 72–74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N₂ atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH^•]_ss which, in turn, leads to an increase in O₂^•- and H₂O₂ mitochondrial production rates.     

Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation

We analyzed the pathogenesis-related generation of H₂O₂ using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H₂O₂ accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H₂O₂ accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H₂O₂ was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H₂O₂ may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.     

Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942

Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO₂ and HCO₃⁻ transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O₂-evolution studies. The K_i for COS inhibition was 29 micromolar for CO₂ transport and 110 micromolar for HCO₃⁻ transport. These results support a model of inorganic carbon transport with a central CO₂ pump and an inducible HCO₃⁻ utilizing accessory protein which supplies CO₂ to the primary pump.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

Responses of marine macroalgae to hydrogen-peroxide stress

In this study, we determined the antioxidative potential of 15 marine macroalgae by measuring the photosynthetic efficiency under artificial oxidative stress after a 30-min exposure to a series of ascending H₂O₂ concentrations. Species exhibiting high maximum quantum yields (F_v/F_m values) were regarded as not susceptible towards H₂O₂ stress. In addition to the short-term stress experiments, the antioxidative defense systems (enzymatic and non-enzymatic) of selected algal species under longer exposure times to H₂O₂ were investigated.Species with striking photosynthetic activity under H₂O₂ stress were Chaetomorpha melagonium (Chlorophyta), showing 40% reduced F_v/F_m as compared to the control after 8 days of exposure to 20 mM H₂O₂. In Fucus distichus (Phaeophyta) F_v/F_m decreased to 50% of the control under the same exposure conditions. Polysiphonia arctica (Rhodophyta) exhibited highest F_v/F_m values with a reduction of only 25%, therefore possessing the highest antioxidative potential of the investigated species.In P. arctica the activities of the antioxidative enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as the pool size of the antioxidant ascorbic acid were investigated. When exposed to different H₂O₂ concentrations (0-2 mM) over 6 days, the intrinsic activities of SOD and GR were stimulated. In a kinetic study over 8 days, the activity of antioxidative enzymes APX and CAT as well as ascorbic acid content were recorded. APX activity was much higher in H₂O₂-treated thalli at the end of the experiment than in the control, also CAT activity increased significantly with increasing H₂O₂ stress. In parallel, ascorbic acid content was reduced under high H₂O₂ concentrations. Furthermore, by using GC-MS techniques in P. arctica bromophenolic compounds with antioxidative properties were identified.This study shows that the measurement of the in vivo fluorescence of photosystem II is a suitable tool to determine the effect of oxidative stress on macroalgae. From these studies it is obvious that different algal species have varying strategies against oxidative stress which correlate with zonation on the shore.     

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H₂O₂) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H₂O₂ was 34.8±2.3% at day 9, and was 70% larger than that without H₂O₂ (21.5±0.8%). Further, H₂O₂ addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H₂O₂, the BrdU assay clearly indicated that H₂O₂ suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H₂O₂ is due to synergistic inhibition of cytokinesis with PMA. Although H₂O₂ did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation.     

Environmental Determinants of Transformation Efficiency in Helicobacter pylori

Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O₂ (5% O₂, 7.6% CO₂, 7.6% H₂) and high O₂ (15% O₂, 2.9% CO₂, 2.9% H₂) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6- to 32-fold greater under high O₂ tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O₂ concentration for these initial experiments also changed the concentrations of CO₂ and H₂, transformations were repeated under conditions where O₂, CO₂, and H₂ were each varied individually. The results showed that the increase in transformation efficiency under high O₂ was largely due to a decrease in CO₂. An increase in pH similar to that caused by low CO₂ was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.