小麦叶片老化期间的内肽酶与H_2O_2的关系(英文)

研究了H2 O2 和蛋白水解酶在小麦 (TriticumaestivumL .cv.Yanmai15 8)叶片老化过程中的关系。小麦叶片老化期间 ,H2 O2 含量高的叶片中内肽酶活力也高。老化后期 ,内源H2 O2 迅速累积 ,内肽酶活力迅速上升 ;通过内肽酶同工酶电泳可检测到新增一种活力较强的内肽酶。用外源H2 O2 处理全展旗叶的内肽酶粗提液 ,随着H2 O2 浓度的升高 ,内肽酶活力先上升后下降。     

在耐热性诱导中豌豆叶片过氧化氢和水杨酸含量与质膜ATP酶活性的变化及相互关系

在高温锻炼(37℃,2h)过程中,豌豆(Pisum sativum L.)叶片过氧化氢(H_2O_2)和游离态水杨酸(SA)含量与质膜ATP酶(H~ -ATPase)活性都有一个高峰,H_2O_2的迸发早于游离态SA的积累,而质膜H~ -ATPase活性高峰的出现则迟于SA高峰;活性氧清除剂、抗氧化剂、质膜NADPH氧化酶抑制剂和H_2O_2的淬灭剂预处理均可有效地阻止高温下H_2O_2和SA的积累以及质膜H~ -ATPase活性的增加。根据以上结果推测,H_2O_2、质膜H~ -ATPase和SA均参与耐热性诱导相关的信号传递,前者作用于SA的上游,而后者在SA下游起作用。     

Ethyl pyruvate-mediated Nrf2 activation and hemeoxygenase 1 induction in astrocytes confer protective effects via autocrine and paracrine mechanisms

Ethyl pyruvate (EP), a simple ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule under various pathological conditions, such as, during cerebral ischemia and sepsis in animal models. Here, the authors investigated the novel molecular mechanism underlying the anti-oxidative effect of EP in primary astrocyte cultures, particularly with respect to nuclear factor E2-related factor 2 (Nrf2) activation and hemeoxygenase 1 (HO-1) induction. EP was found to induce Nrf2 translocation and the inductions of various genes downstream of Nrf2 and these resulted in the amelioration of the oxidative damage of H(2)O(2). Furthermore, EP dose-dependently suppressed H(2)O(2)-induced astrocyte cell death (12h preincubation with 5mM EP increased cell survival after 1h exposure to 100 μM H(2)O(2) from 32.6±0.7% to 63±1.8%). HO-1 was markedly induced (4.9-fold) in EP-treated primary astrocyte cultures and Nrf2 was found to translocate from the cytosol to the nucleus and bind to the antioxidant response element (ARE) located on HO-1 promoter after EP treatment. siRNA-mediated HO-1 or Nrf2 knockdown and zinc protoporphyrin (ZnPP)-mediated inhibition of HO-1 activity showed that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of EP, which was found to involve the ERK and Akt signaling pathways. Furthermore, EP-conditioned astrocyte culture media was found to have neuroprotective effects on primary neuronal cultures exposed to oxidative or excitotoxic stress, and this seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and glutathione (GSH), which accumulated in EP-treated astrocyte culture media. Interestingly, we also found that in addition to HO-1, EP-induced Nrf2 activation increased the expressions of various anti-oxidant genes, including GST, NQO1, and GCLM. The study shows that EP-mediated Nrf2 activation and HO-1 induction in astrocytes act via autocrine and paracrine mechanisms to confer protective effects.     

烟草叶片发育过程中光合功能衰退与H2O2积累的关系

以烟草（Nicotiana tabacum L．cv NC89）为材料,研究了叶片发育过程中H2O2积累与叶绿体光合功能衰退、抗坏血酸-谷胱甘肽（AsA—GSH）循环的关联。结果表明,光合功能衰退过程中,各光合参数均表现为先缓慢后快速的下降趋势,核酮糖-1,5-二磷酸羧化酶（RuBPCase）活性下降较电子传递活性下降迅速,H2O2含量与叶绿素含量、光合速率、RuBPCase活性、抗坏血酸过氧化物酶（APX）、谷胱甘肽还原酶（GR）活性显著负相关。H2O2的定位染色也证实光合功能衰退与H2O2积累密切相关。APX和GR在光合功能可逆衰退阶段维持较高水平,不可逆衰退阶段下降稍快。烟草叶片光合功能衰退快于AsA—GSH循环运转的下调。     

烟草叶片发育过程中光合功能衰退与H_2O_2积累的关系

以烟草(NicotianatabacumL.cvNC89)为材料,研究了叶片发育过程中H2O2积累与叶绿体光合功能衰退、抗坏血酸-谷胱甘肽(AsA-GSH)循环的关联。结果表明,光合功能衰退过程中,各光合参数均表现为先缓慢后快速的下降趋势,核酮糖-1,5-二磷酸羧化酶(RuBPCase)活性下降较电子传递活性下降迅速,H2O2含量与叶绿素含量、光合速率、RuBPCase活性、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)活性显著负相关。H2O2的定位染色也证实光合功能衰退与H2O2积累密切相关。APX和GR在光合功能可逆衰退阶段维持较高水平,不可逆衰退阶段下降稍快。烟草叶片光合功能衰退快于AsA-GSH循环运转的下调。     

Hypoxia regulates PGE(2) release and EP1 receptor expression in osteoblastic cells

Changes in regional O(2) tension that occur during fracture and skeletal unloading may stimulate local bone cell activity and ultimately regulate bone maintenance and repair. The mechanisms by which bone cells sense and respond to changes in O(2) tension are unclear. In this study we investigated the effects of low O(2) on activation of the hypoxia response element (HRE), prostaglandin E(2) (PGE(2)) production, PGE(2) receptor (EP) expression and proliferation in MC3T3-E1 osteoblastic cells. Cells were cultured for up to 72 h in 2% O(2) (considered hypoxic), 5% O(2) (in the range of normal O(2) tension in vivo) or 21% O(2) (commonly used for cell culture). Cells cultured in 2% O(2) showed activation of the HRE, increased PGE(2) release, increased EP1 expression, and reduced cell proliferation compared to cells grown at 21% O(2). Similarly, cells cultured in 5% O(2) showed increased expression of EP1 and a trend toward a decrease in proliferation, but no activation of the HRE or increase in PGE(2) levels. Expression of EP2, EP3 and EP4 were not affected by O(2) tension. The differences in EP receptor profile observed in cells grown at 5% compared to 21% O(2) suggest that bone cell phenotype may be altered under routine cell culture conditions. Furthermore, our data suggest that hypoxia-dependent PGE(2) production and EP1 expression in bone cells may play a role in bone remodeling and repair in regions of compromised or damaged bone, where O(2) tension is low.     

不同老化程度的不结球白菜种子活力指标变化及其相关分析

以不结球白菜品种‘高梗白’种子为材料,采用高温(42℃)高湿(相对湿度100%)人工加速老化处理,研究不同老化程度下种子活力指标的变化及其相关性。结果显示:(1)发芽指标(除不正常苗率)和出苗指标均随种子老化程度的加深而显著下降,不正常苗率显著上升。(2)随老化程度的加深,种子中超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著下降;种子的超氧阴离子(O2.-)产生速率先增高后降低,过氧化氢(H2O2)含量逐渐增加,丙二醛(MDA)含量下降,种子浸出液相对电导率升高;种子的可溶性糖含量、可溶性蛋白含量和脱氢酶活性下降,α-淀粉酶活性先升高后降低。(3)老化种子的发芽势、发芽率、SOD活性、H2O2含量、相对电导率和可溶性糖含量与出苗指标间均存在极显著相关性。研究表明,不结球白菜种子的发芽势、发芽率、SOD活性、H2O2含量和可溶性糖含量随老化程度加深的变化规律一致,且指标间以及与出苗指标均有极显著相关性,可以作为检验不结球白菜种子活力的候选指标。     

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).     

过氧化氢预处理对H9c2心肌细胞氧化应激损伤的保护作用

目的：活性氧介导的氧化损伤是缺血再灌注损伤的重要机制,本研究通过观察H2O2预处理对氧化损伤的H9c2心肌细胞存活率和细胞凋亡的影响,探讨其保护H9c2心肌细胞的作用机制。方法：体外培养H9c2心肌细胞,取对数生长期细胞用于实验研究。建立H2O2预处理抵抗高浓度H：O：诱导的细胞氧化损伤模型,实验分组如下：（1）正常对照组（CTL）;（2）损伤组（INJURY）;（3）预处理组十损伤组（PC）。应用CCK8法检测细胞存活率;试剂盒检测胞内MDA水平和T．sOD活性;Hoechst33258染色观察凋亡形态;Annexin-V／PI双染与流式细胞术检测细胞凋亡率。结果：25vLmol／L的H202预处理90rain能明显地保护H9c2心肌细胞抵抗400μmol／LH2O2诱导的氧化损伤,提高细胞存活率,下调MDA水平,上调SOD活性,抑制细胞凋亡,降低细胞凋亡率。结论：低浓度H2O2预处理能减轻H9c2心肌细胞的氧化损伤,抑制氧化损伤诱导的心肌细胞凋亡,具有很好的抗氧化损伤和抗心肌细胞凋亡的保护作用,其作用机制可能与细胞SOD活性上调有关。H2O2预处理为临床治疗心肌缺血／再灌注损伤提供了一项新策略。     

水分胁迫下苹果叶片的H_2O_2代谢

轻度水分胁迫下苹果叶片Pr迅速升高 ,CAT活性变化不大 ,NaHSO3 处理能显著降低叶内H2 O2 含量 ,表明光呼吸的加强促进了H2 O2 产生可能是叶内H2 O2大量积累的主要原因 ;中度水分胁迫下叶片AsA含量的下降和Mehler反应的增强都非常明显 ,DDTC和AsA处理都能有效降低叶内H2 O2 积累 ,但MV处理的作用不大 ,说明叶片H2 O2 主要来源于Mehler反应 ,AsA降解造成叶片对H2 O2 清除能力的下降是其积累的根本原因 ;严重水分胁迫时 ,NaHSO3 和DDTC都不能有效地减轻叶内H2 O2 积累 ,光呼吸和Mehler反应都可能不是H2 O2 的主要来源     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.     

UV-B辐射引起的绿豆幼苗叶片Rubisco量降低与H2O2含量升高有关

为了探讨UV-B辐射引起的Rubisco含量降低的可能机制,研究了两个绿豆品种(秦豆-20和中绿-1)幼苗在UV-B辐射下叶片Rubisco含量、蛋白水解酶活性和H2O2含量的变化.结果表明:UV-B辐射显著加速了两个绿豆品种幼苗叶片H2O2含量和蛋白水解酶活性上升,使Rubiscco含量下降.秦豆-20品种在UV-B辐射下H2O2含量和蛋白水解酶活性的上升程度明显大于中绿-1,相应其Rubisco含量的下降程度也大于中绿-1.抗坏血酸处理能明显降低UV-B辐射下两品种幼苗叶片H2O2含量,同时明显抑制蛋白水解酶活性的上升及Rubisco含量的下降.结果说明UV-B辐射诱导Rubisco含量的降低可能通过提高H2O2水平从而加强蛋白水解酶系统的活化而加速了Rubisco的降解.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation: NADH oxidation by acetoacetyl-CoA and H2O2

To clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of beta-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the beta-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicating that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA beta-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA beta-oxidation system from the deleterious action of H2O2.     

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

Measurement of H2O2 within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H(2)O(2)) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H(2)O(2) in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H(2)O(2) levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H(2)O(2) to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H(2)O(2) that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H(2)O(2) with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H(2)O(2) correlates with aging, it may not be causative.     

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.     

Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat beta-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca(2+)-permeable channel that is activated by H(2)O(2), and the Ca(2+) influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat beta-cell line RIN-5F. In both cell types, H(2)O(2) induced Ca(2+) influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO(4) and H(2)O(2), to the cells, did not increase intracellular Ca(2+) concentration. Interestingly, when H(2)O(2) was added to the cells under intracellular Fe(2+)-accumulated conditions, Ca(2+) influx was markedly enhanced compared to H(2)O(2) alone. In addition, the H(2)O(2)-induced Ca(2+) influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe(2+)-accumulated conditions, H(2)O(2)-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H(2)O(2). These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H(2)O(2) treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H(2)O(2)-induced cell death in the beta-cell line RIN-5F.     

Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24h after UVB light with only one minimal erythemal dose. Since UVB generates also H(2)O(2), we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1x10(-3)M), the V(max) for enzyme activity increased 4-fold by H(2)O(2) (>2.0x10(-3)M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp(120) residue to 5-OH-Trp(120) by H(2)O(2) causing a conformational change in the regulatory domain. PAH was still activated by H(2)O(2) in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H(2)O(2) by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis.