Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

Oxygen-dependent H2O2 production by Rubisco

Oxygen and ribulose-1,5-bisphosphate dependent, H(2)O(2) production was observed with several wild type Rubisco enzymes using a sensitive assay. H(2)O(2) and d-glycero-2,3-pentodiulose-1,5-bisphosphate, a known and potent inhibitor of Rubisco activity, are predicted products arising from elimination of H(2)O(2) from a peroxyketone intermediate, specific to oxygenase activity. Parallel assays using varying CO(2) and O(2) concentrations revealed that the partitioning to H(2)O(2) during O(2) consumption by spinach Rubisco was constant at 1/260-1/270. High temperature (38 degrees C), which reduces Rubisco specificity for CO(2) versus O(2), increased the rates of H(2)O(2) production and O(2) consumption, resulting in a small increase in partitioning to H(2)O(2) (1/210). Two Rubiscos with lower specificity than spinach exhibited greater partitioning to H(2)O(2) during catalysis: Chlamydomonas reinhardtii (1/200); and Rhodospirillum rubrum (1/150).     

2H magnetic relaxation of 2H2O absorbed by wool.

D2O absorbed by intact wool fibers was studied by solid-state 2H nuclear magnetic resonance (NMR) spectroscopy. In wool fibers swollen in D2O, the deuteron transverse magnetization and the spin-locked magnetization revealed a non-exponential decay. At least two NMR phases with different sets of the NMR relaxation parameters, T(1rho) (2H) and T2 2H, have been detected that may be a manifestation of two different morphological phases of the cortex of the fiber.     

微胶囊固定化过氧化氢酶的制取及对H_2O_2的分解作用

以乙基纤维素为膜材,用溶液干燥法制取了微胶囊固定化过氧化氢酶,探讨了制取过程中明胶的加入对微胶囊产率及固定化过氧化氢酶活性的影响,同时论述了存放时间、温度以及环境ｐＨ值对微胶囊固定化过氧化氢酶稳定性的影响．深入研究了微胶囊固定化过氧化氢酶对Ｈ２Ｏ２的分解作用,获得了十分有意义的结果     

Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.     

Hyperoxia increases H2O2 production by brain in vivo

Hyperoxia and hyperbaric hyperoxia increased the rate of cerebral hydrogen peroxide (H2O2) production in unanesthetized rats in vivo, as measured by the H2O2-mediated inactivation of endogenous catalase activity following injection of 3-amino-1,2,4-triazole. Brain catalase activity in rats breathing air (0.2 ATA O2) decreased to 75, 61, and 40% of controls due to endogenous H2O2 production at 30, 60, and 120 min, respectively, after intraperitoneal injection of 3-amino-1,2,4-triazole. The rate of catalase inactivation increased linearly in rats exposed to 0.6 ATA O2 (3 ATA air), 1.0 ATA O2 (normobaric 100% O2) and 3.0 ATA O2 (3 ATA 100% O2) compared with 0.2 ATA O2 (room air). Catalase inactivation was prevented by pretreatment of rats with ethanol (4 g/kg), a competitive substrate for the reactive catalase-H2O2 intermediate, compound I. This confirmed that catalase inactivation by 3-amino-1,2,4-triazole was due to formation of the catalase-H2O2 intermediate, compound I. The linear rate of catalase inactivation allows estimates of the average steady-state H2O2 concentration within brain peroxisomes to be calculated from the formula: [H2O2] = 6.6 pM + 5.6 ATA-1 X pM X [O2], where [O2] is the concentration of oxygen in ATA that the rats are breathing. Thus the H2O2 concentration in brains of rats exposed to room air is calculated to be about 7.7 pM, rises 60% when O2 tension is increased to 100% O2, and increases 300% at 3 ATA 100% O2, where symptoms of central nervous system toxicity first become apparent. These studies support the concept that H2O2 is an important mediator of O2-induced injury to the central nervous system.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O

H₂–forming N ⁵,N ¹⁰ –methylenetetrahydromethanopterin dehydrogenase is a novel type of hydrogenase that contains neither nickel nor iron-sulfur clusters. Evidence has been presented that the reaction mechanism catalyzed by the enzyme is very similar to that of the formation of carbocations and H₂ from alkanes under superacidic conditions. We present here further results in support of this mechanism. It was found that the purified enzyme per se did not catalyze the conversion of para H₂ to ortho H₂, a reaction catalyzed by all other hydrogenases known to date. However, it catalyzed the conversion in the presence of the substrate N ⁵,N ¹⁰ –methenyltetrahydromethanopterin (CH≡H₄MPT⁺), indicating that for heterolytic cleavage of H₂ the enzyme-CH≡H₄MPT⁺ complex is required. In D₂O, the formation of HD and D₂ from H₂ rather than a para–ortho H₂ conversion was observed, indicating that after heterolytic cleavage of H₂ the dissociation of the proton from the enzyme-substrate complex is fast relative to the re-formation of free H₂.     

黄芪注射液对L6成肌细胞抗H2O2致凋亡模型的保护

目的:探讨黄芪注射液对H2O2损伤L6大鼠成肌细胞的修复.方法:取1、0.5、0.1、0.05mmol/L H2O2损伤成肌细胞建立模型,再取2 000、1 000、500、250、125、62.5mg/mL黄芪注射液实施保护.应用MTT法、流式细胞仪及荧光抗体Bax和Bcl-2检测,使用SPSS 15.0统计软件处理数据.结果:经0.1mmol/L H2O2损伤成肌细胞存活率由94.4±5.7％降至32.6±3.8％,凋亡率由0增至32.45±2.9％.黄芪注射液处理实验组细胞存活率64.5±4.8 ～82.2±9.6％较模型组32.6±3.8％增加,凋亡率也从32.45±2.9％减至2.96±0.5 ～9.29±2.7％,荧光抗体检测Bcl-2细胞数57.7±4.3～74.2±6.9,Bax细胞数42.3±4.0～60.2±5.1.结论:黄芪注射液对大鼠成肌细胞H2O2损伤经由p38MAPK信号通路实施保护.     

Fourier transform infrared studies of ribonuclease in H2O and 2H2O solutions

Fourier transform infrared transmission spectra have been obtained of the enzyme ribonuclease in both H₂O and ²H₂O. The resolution of the spectra have been enhanced by Fourier self-deconvolution procedures. The infrared spectrum of ribonuclease changes during exchange of the enzyme's amide hydrogens for deuterium and the exchange has been followed in the amide I and amide II spectral regions. The amide I band shifts towards lower wavenumbers during both the fast and slow phases of hydrogen exchange and the interpretation of these shifts has aided the band assignments. In particular these studies have allowed an assignment to be made for the high frequency component of the β-strand absorption that differs from that proposed previously. This paper represents the first example of the use of deconvoluted Fourier transform infrared spectra in conjunction with hydrogen-deuterium exchange in order to aid in the assignment of a proteins's infrared bands.     

Nitroxide-Stimulated H2O2 Decomposition by Peroxidases and Pseudoperoxidases

Many copper and iron complexes can be reduced by O^-₂ as well as by H₂O₂. According to the rates of reduction and the concentration of O^-₂ and H₂O₂, the metal complexes may serve either as catalyst of O^-₂ dismutation or as catalysts of the reaction between O^-₂ and H₂O₂ to form OH' radical (Haber-Weiss reaction). Various factors which influence whether metal complexes protect the biological systems from superoxide toxicity or enhance it are discussed.     

Decomposition of H2O2 by human cataractous lenses

It was shown that human lens opacity was accompanied by the decrease in the lens ability to cleave H2O2 (10(-4) M), added to the lens-surrounding medium. The rate of peroxide decomposition at the stage of mature cataract in isolated human lenses was 3.5 times lower than that of the control human lenses (transparent lens, initial cataract). Specific catalase inhibitor--3-amino,IH-1,2,4-triazole showed no significant influence on the rate of H2O2 cleavage. Reduced glutathione (10 microM) added to the lens incubation medium induced a sharp increase in the rate of H2O2 detoxication. The results indicate that reduced glutathione metabolism is of primary importance in the maintenance of anti-peroxide activity in the lens.     

H2O2-Induced Inhibition of Photosynthetic O2 Evolution by Anabaena variabilis Cells

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.     

Nitroxide-Stimulated H2O2 Decomposition by Peroxidases and Pseudoperoxidases

Nitroxide free radicals interact with Hb/metHb, Mb/metMb and with peroxidases/phenols to induce a catalase-like conversion of H₂O₂ to O₂ (catalatic activity), without being substantially consumed in the process. The mechanism of this reaction is postulated to involve a one-electron oxidation of the nitroxide to the immonium oxene, which then reacts further to release oxygen and the nitroxide. An involvement of the immonium oxene in the reaction mechanism is consistent with ferryl heme reduction by nitroxides and a detection of the reduced nitroxide when the reaction mixture is supplemented with the two-electron reductant sodium borohydride. The nitroxide-induced catalatic activity is completely inhibited when the reaction mixture is supplemented with glutathione. Nitroxides suppress free radical formation by hydroperoxide-activated heme proteins, as inferred from their inhibition of the spin-trapping of glutathionyl radicals. H₂O₂ decomposition and a suppression of reactive free radical formation by heme proteins appears to be an antioxidant activity of nitroxides, which is distinct from their previously reported superoxide dismutating activity and which may be a factor in their protective action in models of cardiac reperfusion injury.     

Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.     

Diverse catalytic activities in the alphabeta-hydrolase family of enzymes: activation of H2O, HCN, H2O2, and O2

The article describes the observation of novel catalytic activities in the alphabeta-hydrolase superfamily apparently unrelated to ester hydrolysis and unexpected biochemical observations relating to the structure and function of the serine catalytic triad in these enzymes. One common feature of these novel activities is the activation of a small diatomic molecule, but via diverse chemistry. Possible mechanisms of catalysis are discussed.     

Mechanism of H2O2 production in porcine thyroid cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H2O2-generating machinery.

Hydrogen peroxide (H2O2), which is required for thyroid hormone synthesis, has been believed to be produced at the apical cell surface of thyroid follicular cells. However, we recently found that plasma membrane from porcine thyroid exclusively generated superoxide anion (O2-) by employing a novel method for simultaneous determination of H2O2 and O2- with diacetyldeuterioheme-substituted horseradish peroxidase (diacetyl-HRP) as the trapping reagent [Nakamura, Y., Ohtaki, S., Makino, R., Tanaka, T., & Ishimura, Y. (1989) J. Biol. Chem. 264, 4759-4761]. The present study describes the mechanism of H2O2 production as analyzed by this new method. Incubation of cultured porcine follicular cells with ionomycin, a Ca-ionophore, caused an increase in oxygen uptake of about 80%. During enhanced respiration, the cells released H2O2 in an amount equivalent to the amount of oxygen consumed as judged by the formation of compound II of diacetyl-HRP, and H2O2 adduct of the peroxidase. No formation of compound III of the peroxidase, an O2- adduct, was detected during burst respiration. Thus, the intact cells exclusively released H2O2 to the outside of the cells. On the other hand, when the cell fragments from follicular cells were incubated with NADPH or NADH in the presence of Ca2+, the production of O2- was observed only during NADPH-dependent burst respiration, supporting our previous results that the plasma membrane exhibited NADPH-dependent O2(-)-generating activity. O2- production by the plasma membrane was further confirmed by analyses of the effects of superoxide dismutase (SOD) and catalase on the reaction. These results suggested that H2O2 is secondarily produced through the dismutation of O2-.(ABSTRACT TRUNCATED AT 250 WORDS)     

藻类产生及清除过氧化氢的研究

过氧化氢的生物生成是天然水体中H2O2的来源之一。从藻类产生及分解过氧化氢的途径,影响过氧化氢产量的主要因素,如藻的种类、细胞的渗透性、藻的生长阶段、藻浓度和光照条件等几方面对这一领域的研究作了综述。     

Regulation of Bcl-xL expression by H2O2 in cardiac myocytes

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.     

H2O2 and (.)NO scavenging by Mycobacterium leprae truncated hemoglobin O

Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H₂O₂ and of trHbOFe(IV)O reduction by NO and NO₂⁻ are reported. The value of the second-order rate constant for H₂O₂-mediated oxidation of trHbOFe(III) is 2.4 × 10³ M⁻¹ s⁻¹. The value of the second-order rate constant for NO-mediated reduction of trHbOFe(IV)O is 7.8 × 10⁶ M⁻¹ s⁻¹. The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 × 10¹ s⁻¹. The value of the second-order rate constant for NO₂⁻-mediated reduction of trHbOFe(IV)O is 3.1 × 10³ M⁻¹ s⁻¹. As a whole, trHbOFe(IV)O, generated upon reaction with H₂O₂, catalyzes NO reduction to NO₂⁻. In turn, NO and NO₂⁻ act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H₂O₂ and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration.