柠檬酸铁对亚硝酸根硝化酪氨酸反应的影响

由一氧化氮和超氧阴离子迅速反应发生的过亚硝酸根（ＯＮＯＯ＾－）是一种强细胞毒性物质。使含酚基物质如酪氨酸等硝化,是过亚硝酸根损伤生物系统的重要途径之一。研究了柠檬酸铁和草酸铁对过亚硝酸根硝化酪氨酸反应的影响。在生理ｐＨ条件下柠檬酸铁和草酸铁对硝化反应无影响。在弱酸性条件下柠檬酸铁和草酸铁可催化硝化反应,对ｐＨ影响配合物在硝化反应中的催化活性的原因进行了讨论。     

过氧亚硝酸根与细胞信号转导

生物系统中产生的过氧亚硝酸根(peroxynitrite,ONOO-)具有强氧化性,能够损伤多种生物大分子,产生细胞毒性。细胞通过激活信号通路产生应激反应,其中包括蛋白质酪氨酸激酶(PTK)依赖的多种路径,而ONOO-通过硝化或氧化作用调节酪氨酸的磷酸化。酪氨酸残基的硝化能直接影响酪氨酸的磷酸化,而磷酸酶的氧化将导致酪氨酸磷酸化/去磷酸化平衡的改变,ONOO-激活细胞信号转导通路的作用机制对认识其生理病理功能具有重要意义。     

铁过载促进小鼠肝组织发生蛋白质酪氨酸硝化

蛋白质酪氨酸硝化是一种蛋白质翻译后的修饰,其存在会影响酶的催化活性,细胞信号转导和细胞骨架结构．在铁过载情况下,存在引起蛋白质酪氨酸硝化的有利环境,但目前尚无实验证实．本文运用腹腔注射右旋糖苷铁造成小鼠铁过载模型,通过免疫印迹法发现,在铁过载情况下,肝中诱导型一氧化氮合酶表达显著高于正常对照小鼠;铁过载小鼠肝中总体蛋白质硝化程度高于正常小鼠;铁过载引起的蛋白质酪氨酸硝化有一定的选择性,在铁过载小鼠肝中发现一些新的被硝化蛋白质条带（约 57 kD、 35 kD）．上述结果证实,铁过载会促进肝蛋白质酪氨酸硝化．     

蛋白质酪氨酸硝化和细胞信号转导

蛋白质硝基酪氨酸作为一氧化氮(NO)衍生的蛋白质翻译后修饰产物,被认为是许多生理和病理条件下的生物标志物。本文综述了蛋白质酪氨酸硝化可以作为信号调节元件与已知的信号途径相关,包括NO、蛋白质酪氨酸激酶、丝裂原激活蛋白激酶、T-淋巴细胞、转录因子NF-κB、Ca2 等。同时也论证了蛋白质酪氨酸硝化作为信号转导元件的可能性。     

蛋白质硝基化反应的途径

导致蛋白质发生硝基化反应的途径有多种,主要可以分为ONOO^-途径和非ONOO^-途径两类。ONOO^-途径导致的蛋白质硝基化可因金属离子或CO2的存在而强化,非ONOO^-途径导致的蛋白质硝基化则是亚硝酸盐或其它含氮物质在氧化剂存在下,经含铁卟啉的蛋白质催化而引发的。各种途径导致的蛋白质硝基化都伴随着蛋白质氧化的存在。     

草酸对黄瓜根中铁还原的促进作用

用黄瓜为材料,研究了草酸对植物根切段还原Ｆｅ（Ⅲ）ＥＤＴＡ的促进作用。在２－１４ｍｍｏｌ／Ｌ范围内随着草酸浓度的加大,其促进作用不断提高;在４ｈ内随着反应时间的推移,Ｆｅ（Ⅲ）ＥＤＴＡ的还原量成线性上升趋势。进一步用完整根、粗酶提取液和提纯的质膜证明：促进作用并非草酸本身作为电子供体直接或间接地加速了铁还原反应,而是形成的草酸铁螯合态是根中铁还原酶更有效的底物。整体根还原草酸铁的活力和质膜铁还原酶     

蚯蚓和铁处理对苹果根铁营养影响

试验于2007～2009年在河北省永年县曹庄村和中国农业大学曲周实验站进行。在苹果树根际用不同价态的2500、5000、10000、20000mg/kg的铁处理玉米秸秆后接种蚯蚓,研究蚯蚓和铁对苹果根系生长、蚯蚓对铁的富集转移及根质外体铁的影响。结果表明:蚯蚓对铁有很大的富集量,在20000mg/kg(试验所用最高浓度)二价铁和三价铁处理的秸秆中可以成活并把秸秆转化为蚯蚓粪,促进根系生长,提高根的质外体铁含量,蚯蚓对二价铁的适应性高于三价铁。蚯蚓可将有机物料中的铁转移到果树根系内,5000mg/kg铁处理增加蚯蚓体内Fe2+含量和根质外体铁含量效果最好,蚯蚓、蚯蚓粪和根中的全铁含量随铁处理浓度的增加而增加。铁显著促进果树根系生长,没有用铁处理过的秸秆接种蚯蚓诱导的新根量明显少于用铁处理的,两种不同价态的铁都是以5000mg/kg的新根量最多。蚯蚓显著促进根系生长,没有接种蚯蚓的处理新根量显著少于接种蚯蚓的处理。     

草酸对黄瓜根中铁还原的促进作用

用黄瓜为材料 ,研究了草酸对植物根切段还原Fe(Ⅲ )EDTA的促进作用。在 2～ 14mmol/L范围内随着草酸浓度的加大 ,其促进作用不断提高 ;在 4h内随着反应时间的推移 ,Fe(Ⅲ )EDTA的还原量成线性上升趋势。进一步用完整根、粗酶提取液和提纯的质膜证明 :促进作用并非草酸本身作为电子供体直接或间接地加速了铁还原反应 ,而是形成的草酸铁螯合态是根中铁还原酶更有效的底物。整体根还原草酸铁的活力和质膜铁还原酶催化草酸铁的效率 (Vmax/Km)都远大于还原柠檬酸铁和Fe (Ⅲ )EDTA的活力和效率     

柠檬酸和草酸对Pb胁迫下马蔺生长和生理的影响

采用营养液培养方法,研究了外源柠檬酸(0.5、5mmol.L-1)和草酸(0.5、5mmol.L-1)对不同浓度Pb(0、100、500mg.L-1)胁迫下马蔺(Irislactea var.chinensis)生长和生理的影响。结果表明,与单独Pb胁迫相比,加入5mmol.L-1的柠檬酸显著促进了马蔺株高的生长;草酸的加入缓解了500mg.L-1Pb胁迫对马蔺根生长的影响;加入0.5mmol.L-1的草酸缓解了500mg.L-1Pb胁迫对马蔺地上部生长的影响,并显著增加了100和500mg.L-1Pb胁迫下马蔺的地下部干质量;0.5mmol.L-1的柠檬酸能显著增加100mg.L-1Pb胁迫下马蔺叶片SOD酶活性;0.5和5mmol.L-1柠檬酸和0.5mmol.L-1草酸均能显著增加100mg.L-1Pb胁迫下马蔺体内POD酶活性;5mmol.L-1的柠檬酸和0.5mmol.L-1的草酸均缓解了500mg.L-1Pb胁迫对马蔺光合色素的影响;0.5和5mmol.L-1的柠檬酸和0.5mmol.L-1的草酸均能缓解由100和500mg.L-1Pb胁迫引起马蔺叶片细胞膜的损伤。     

Protein tyrosine nitration in cellular signal transduction pathways

How specificity and reversibility in tyrosine nitration are defined biologically in cellular systems is poorly understood. As more investigations identify proteins involved in cell regulatory pathways in which only a small fraction of that protein pool is modified by nitration to affect cell function, the mechanisms of biological specificity and reversal should come into focus. In this review experimental evidence has been summarized to suggest that tyrosine nitration is a highly selective modification and under certain physiological conditions fulfills the criteria of a physiologically relevant signal. It can be specific, reversible, occurs on a physiological time scale, and, depending on a target, can result in either activation or inhibition.     

Nitrotyrosine as a marker for peroxynitrite‐induced neurotoxicity: the beginning or the end of the end of dopamine neurons?

This review examines the involvement of nitrotyrosine as a marker for peroxynitrite-mediated damage in the dopamine neuronal system. We propose that the dopamine neuronal phenotype can influence the cytotoxic signature of peroxynitrite. Dopamine and tetrahydrobiopterin are concentrated in dopamine neurons, and both are essential for their proper neurochemical function. It is not well appreciated that dopamine and tetrahydrobiopterin are also powerful blockers of peroxynitrite-induced tyrosine nitration. What is more, the reaction of peroxynitrite with either dopamine or tetrahydrobiopterin forms chemical species (i.e. o-quinones and pterin radicals, respectively) whose cytotoxic effects may be manifested far earlier than nitrotyrosine formation in the course of dopamine neuronal damage. A better understanding of how the dopamine neuronal phenotype modulates the effects of reactive nitrogen species could reveal early steps in drug- and disease-induced damage to the dopamine neuron and form the basis for rational, protective therapies.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

Consequences of MnSOD interactions with nitric oxide: nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^- ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^-  species initiate the formation of peroxynitrite (ONOO^- ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^-  decomposition and ONOO^- -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^-  is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Efficiency of methemoglobin, hemin and ferric citrate in catalyzing protein tyrosine nitration, protein oxidation and lipid peroxidation in a bovine serum albumin-liposome system: Influence of pH

Protein tyrosine nitration, protein oxidation and lipid peroxidation are nitrative/oxidative modification of protein and lipids. In this paper, a BSA (bovine serum albumin)-lecithin liposome system was used to study the nature of different forms of iron, including methemoglobin, hemin and ferric citrate, in catalyzing H₂O₂-nitrite system to oxidize protein and lipid as well as nitrate protein. It was found that in pH range of 5.0-9.0, in pure BSA solution or pure liposome solution, hemin and methemoglobin catalyzed protein tyrosine nitration and lipid peroxidation were decreased with the increasing of pH, while hemin and methemoglobin catalyzed protein oxidation was significantly and moderately increased, respectively. Lipid completely inhibited hemin catalyzed protein tyrosine nitration but only partially inhibited methemoglobin catalyzed protein tyrosine nitration, and its inhibitory effect on hemin induced protein oxidation was also more pronounced. In addition, BSA showed more efficient in inhibiting hemin and ferric citrate induced lipid peroxidation. At the same condition, ferric citrate was relatively ineffective in all tests. Considering protein tyrosine nitration, protein oxidation and lipid oxidation as overall oxidative damage, these results indicated that methemoglobin is more toxic than hemin and ferric citrate, the degradation procedure of heme containing macromolecules, e.g. hemoglobin to hemin and finally to low molecular weight bounded iron, is step by step detoxification. These results provide fundamental knowledge on oxidative/nitrative of biomolecules in lipid-protein coexistence system.     

Formation of Nitrated and Hydroxylated Aromatic Compounds from Benzene and Peroxynitrite, A Possible Mechanism of Benzene Genotoxicity

Peroxynitrite, the reaction product of nitric oxide (NO*) and superoxide anion (O*-) produced during immune activation by a variety of inflammatory cells, may contribute to genotoxicity of benzene through its ability to carry out hydroxylation and nitration. After exposure of benzene to synthesised peroxynitrite, phenol, nitrophenols (p-nitrophenol, o-nitrophenol and m-nitrophenol) and nitrobenzene were identified in the reaction mixture by HPLC separation and single UV wavelength and diode array detection. The formation of phenol, nitrophenols and nitrobenzene showed a linear relationship with both benzene and peroxynitrite concentrations. The molar ratio for phenol/(nitrobenzene and nitrophenols) was approximately 9/5 with a total product yield of 14% hydroxylated and nitrated products as based on peroxynitrite. The physiological relevance of the chemical reaction between benzene and peroxynitrite was tested by detecting the reaction products in human neutrophils (2.5 ± 10⁷ cells/ml) incubated with 10 mM benzene for 25 min. The concentration of phenol and p-nitrophenol were found to be 1.29 ± 0.22 and 1.56 ± 0.61 μM mean ± SD) in the incubation medium of the neutrophils pretreated with phorbol myristate acetate (500 nM) for 5 min, respectively, whereas no metabolites were detected if the neutrophils were not pretreated. Nitrated aromatic compounds are known to be more carcinogenic than the parent compounds. It is reported that acute and chronic infection increases the risk of cancer at various sites; and that anti-inflammatory agents decrease benzene myelotoxicity. We suggest that the increased production of peroxynitrite during chronic inflammation combined with benzene exposure may increase the carcinogenicity of benzene by a mechanism that includes the formation of metabolites from the chemical reaction between benzene and peroxynitrite. Thus, peroxynitrite mediated hydroxylation and nitration of benzene during immune activation represent a novel in vivo mechanism for generation of proximal carcinogens of benzene.     

Protein nitration

Various proteins/enzymes obtained commercially were tested for the presence of endogenously nitrated tyrosine by Western blot analysis omitting reducing agent in the step of SDS-PAGE. Histones II-S and VIII-S, IgG, cAMP-dependent protein kinase (PKA), phosphorylase b, and phosphorylase kinase exhibited strong immunoreactive bands. Histone VI-S, glycogen synthase, lactate dehydrogenase, actin, thyroglobulin, and macroglobulin exhibited moderate immunoreactivity. Histone III-S, casein, acetyl cholinesterase, DNase I, and lipase had only traceable immunoreactivity. Whereas histone VII-S, pyruvate kinase, trypsin, pepsin, chymotrypsin, protease IV, and protease XIII, and glutathione S-transferase lacked immunoreactivity. A variation of immunoreactivity between hypertensive and normaltensive rat hearts was found in the histone-agarose fractions of crude extracts. Additionally, nitrotyrosine immunoreactivity was observed in non-mammalian organisms including Eschericia coli, Saccharomyces cerevisiae and Triticum vulgaris. Upon the treatment of 15 M peroxynitrite (PN), strong oxidant derived from nitric oxide (NO), the apparent K_m of PKA for cAMP increased from approximately 10^-8 to 10^-6 M. The results imply that the varied nitration of tyrosine residues in proteins/enzymes may occur as a post-translational modification in vivo, and such discriminative nitration may be vital in PN/NO-regulated signal transduction cascade.     

黄瓜叶片对草酸铁的还原作用

铁还原作用在植物叶片对铁素吸收及利用过程中起关键作用.本研究表明相对于其它几种常用的铁螯合物如二乙基四乙酸铁(FeⅢEDTA)或柠檬酸铁,草酸铁更有利于黄瓜活体叶片及铁还原酶的作用,即表现出更高的铁还原活力.缺铁降低了黄瓜叶片中的铁还原活性.缺铁时叶片中的草酸含量不受影响,而富含在石灰性缺铁土壤中的碳酸氢根离子能使叶片中草酸含量显著提高.