用N-3芘NEM标记巯基研究高铁卟啉化合物对红细胞膜蛋白的影响

N-(3芘)马来酰亚胺(N-3芘NEM)是一种特异标记蛋白质巯基的荧光探针,可通过荧光强度测定巯基含量。本文用不同浓度的高铁卟啉处理红细胞膜,再用N-3芘NEM标记,发现随浓度加大,巯基含量减少,与化学法测定结果一致,说明高铁卟啉可使膜蛋白巯基交联。用SDS-聚丙烯酰胺凝胶电泳法做N-3芘NEM标记的红细胞膜,通过荧光扫描确定N-3芘NEM与红细胞膜区带1、2及3结合。     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

膜脂状态在Mg~2+对线粒体H~+-ATP酶影响中的作用

对猪心线粒体H-ATP酶体系中膜脂在Mg~(2 )的激活功能中起极重要的作用。通过DPH外源荧光探针和5-NSESR探针测膜脂表层Mg~(2 )作用影响的研究表明Mg~(2 )首先对膜脂作用,增加膜脂的有序性。TU颗粒和复合体的酶、色氨酸残基内源荧光偏振度测定的结果表明Mg~(2 )可进而影响内嵌蛋Fo,最终导致F_1上功能位点的构象和功能的变化。但膜脂的原始状态对Mg~(2 )起作用是重要的。     

用激光衍射法研究红细胞在低粘切变流场中的取向和变形及其生物物理意义

采用一种新型激光衍射法 ,对不同浓度WGA处理的红细胞进行测量 ,进一步研究了红细胞在低粘切变流场中的流变特性 .通过用DPH探剂标记的荧光偏振法 ,用马来酰亚胺 (MSL)标记的电子顺磁共振法 ,揭示了新激光衍射法所描写的红细胞在低粘切变流场中取向指数 (DI_or)与等容小变形指数 (DI_d)的生物物理意义     

硒与巯基作用稳定红细胞膜骨架

Na_2SeO_3可以减少人红细胞膜血影收缩蛋白(Spectrin)从膜骨架上解离下来.当含有巯基的二硫苏糖醇(DTT)存在下或红细胞膜用碘代乙酰胺(IAA)封闭巯基后.Na_2SeO_3的效应就不再呈现.用与巯基结合的荧光探针:N-[3-芘]-马来酰胺(N-[3-P]-M)来测试不同浓度Na_2SeO_3存在下红细胞膜的荧光强度.结果表明,随着Na_2SeO_3浓度增加,荧光强度就相应降低,这都说明硒是通过与红细胞膜巯基相作用来发挥效应的.~(31)P-NMR测试表明,人红细胞膜在加硒条件下透析,化学位移各向异性(Δσ)值低于不加硒的样品,这提示硒与红细胞膜蛋白巯基作用后红细胞膜脂-蛋白质的相互作用发生了变化.根据实验结果对硒作用后可能引起膜蛋白构象的变化也进行了讨论.     

锌_7-与镉_7-金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量．结果表明,锌7-与镉7-金属硫蛋白对羟自由基引起的膜脂流动性减低、脂质过氧化反应增强双膜蛋白构象改变有明显抑制作用,而且,前者的作用明显强于后者．     

锌7—与镉7—金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量。结果表明,锌７－与镉７－金属硫蛋白对羟自由基引起的膜脂流劝性减低,脂质过氧化反应增强及膜蛋白构象及改变有明显的抑制作用,而且,前的作用明显强于后。     

活性氧所致超氧化物歧化酶肽链断裂的观察

探究活性氧所致铜锌超氧化物歧化酶（ＳＯＤ）肽链断裂的情况,将过氧化氢或抗坏血酸－Ｆｅ（Ⅲ）分另竽马来酰亚胺标记的ＳＯＤ,然后用高效液相反应相色谱（ＲＰ－ＨＰＬＣ）分析,经１ｍｍｏｌ／ＬＨ２Ｏ２处理后ＳＯＤ用ＲＰ－ＨＰＬＣ分离出二个肽段,用顺磁共振检测显示只有一个肽段具有马来酰亚胺信号经５ｍｍｏｌ／ＬＨ２Ｏ２处理后ＳＯＤ有四个肽段生成,其中有一个肽段具有马来酰亚胺信号,用５ｍｍｏｌ／Ｌ抗坏血酸和０．     

硒与红细胞血影收缩蛋白(Spectrin)作用导致构象变化

从人红细胞膜提取血影收缩蛋白(Spectrin),研究不同浓度Na_2SeO_3与其作用后的构象变化.用N—[3—芘]—马来酰胺(N-[3-P]M)作荧光探针标记Spectrin,经SDS处理后,其荧光强度随硒的浓度增加而逐步降低.但未经SDS处理的样品,加入0.2—1.0ppm的Na_2SeO_3后反而使荧光强度有所增加.Spectrin经硒作用与未经作用相比较在色氨酸内源荧光、丹磺酰氯(DNS-Cl)标记后(DNS-Spectrin)的荧光光谱以及色氨酸残基与DNS基团间的能量转移实验结果均有明显的差别.这反映Spectrin的巯基经与硒作用后会导致构象的变化.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

Reactive oxygen injury to cultured pulmonary artery endothelial cells: mediation by poly(ADP-ribose) polymerase activation causing NAD depletion and altered energy balance.

The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (SOD), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl-p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of 3-aminobenzamide (3AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD+ and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either SOD, CAT, or DFX were added at the same time as DHF, suggesting that O2-., H2O2, and HO. mediated these changes. The O2-. appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD+, and the decline in ATP were also prevented by the addition of 3AB. The oxidant-induced DNA strand breakage was, however, unaffected by either 3AB or NA. Addition of 3AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., "rescued" the cells when neither SOD, CAT, nor DFX addition were effective. Concurrent with the "rescue" from lysis by 3AB, there was an increase in NAD+ content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduction in molecular synthesis or enzyme activity of superoxide dismutases and catalase contributes to oxidative stress and neurogenic hypertension in spontaneously hypertensive rats

A balance between production and elimination of reactive oxygen species such as superoxide anion (O2*-) and hydrogen peroxide (H2O2) tightly regulates the homeostasis of cellular oxidative stress, which contributes to a variety of cardiovascular diseases, including hypertension. The present study assessed the hypothesis that O2*- or H2O2 levels augmented by the reduced molecular synthesis or enzyme activity of superoxide dismutase (SOD), catalase (CAT), or glutathione peroxidase (GPx) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that generate tonic vasomotor tone are located, contribute to the pathogenesis of hypertension. We found that copper/zinc SOD (SOD1), manganese SOD (SOD2), or CAT, but not GPx, mRNA or protein expression and enzyme activity in the RVLM of spontaneously hypertensive rats (SHR) were significantly lower than those in normotensive Wistar-Kyoto (WKY) rats, along with a significantly higher level of O2*- or H2O2. A causative relationship between these biochemical correlates of oxidative stress and neurogenic hypertension was established when gene transfer by microinjection of adenovirus encoding SOD1, SOD2, or CAT into the bilateral RVLM promoted a long-lasting reduction in arterial pressure in SHR, but not WKY rats, accompanied by an enhanced SOD1, SOD2, or CAT protein expression or enzyme activity and reduced O2*- or H2O2 level in the RVLM. These results together suggest that downregulation of gene expression and enzyme activity of the antioxidant SOD1, SOD2, or CAT may underlie the augmented levels of O2*- and H2O2 in the RVLM, leading to oxidative stress and hypertension in SHR.     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

Superoxide formed from cigarette smoke impairs polymorphonuclear leukocyte active oxygen generation activity.

Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, O2-. was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated O2-. was an initiating factor in the impairment and OH. generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as alpha-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) O2-. is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated O2-. can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as alpha-tocopherol or DHLA might be successful in preventing these deleterious effects.     

Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defence system and reactive oxygen species production

We investigated the interaction among abscisic acid (ABA), reactive oxygen species (ROS) and antioxidant defence system in the transduction of osmotic stress signalling using Arabidopsis thaliana WT (Columbia ecotype, WT) and an ABA-deficient mutant (aba2-1). For this, 50 μm ABA and osmotic stress, induced with 40% (w/v) polyethylene glycol (PEG8000; -0.7 MPa), were applied to WT and aba2-1 for 6, 12 or 24 h. Time course analysis was undertaken for determination of total/isoenzyme activity of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), NADPH oxidase (NOX; EC 1.6.3.1) activity; scavenging activity of the hydroxyl radical (OH˙), hydrogen peroxide (H(2) O(2) ); endogenous ABA and malondialdehyde (MDA). The highest H(2) O(2) and MDA content was found in PEG-treated groups of both genotypes, but with more in aba2-1. ABA treatment under stress reduced the accumulation of H(2) O(2) and MDA, while it promoted activity of SOD, CAT and APX. APX activity was higher than CAT activity in ABA-treated WT and aba2-1, indicating a protective role of APX rather than CAT during osmotic stress-induced oxidative damage. Treatment with ABA also significantly induced increased NOX activity. Oxidative damage was lower in ABA-treated seedlings of both genotypes, which was associated with greater activity of SOD (Mn-SOD1 and 2 and Fe-SOD isoenzymes), CAT and APX in these seedlings after 24 h of stress. These results suggest that osmotic stress effects were overcome by ABA treatment because of increased SOD, CAT, APX and NOX.     

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endothelium-denuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.     

Different drought-stress responses in photosynthesis and reactive oxygen metabolism between autotetraploid and diploid rice

Photosynthetic light curve, chlorophyll (Chl) content, Chl fluorescence parameters, malondialdehyde (MDA) content, phosphoenolpyruvate carboxylase (PEPC) activity and reactive oxygen metabolism were studied under drought stress in two autotetraploid rice lines and corresponding diploid rice lines. Net photosynthetic rate decreased dramatically, especially under severe drought stress and under high photosynthetic active radiation in diploid rice, while it declined less under the same conditions in autotetraploid lines. Compared with the corresponding diploid lines, the Chl content, maximum photochemical efficiency of photosystem (PS) II, and actual photochemical efficiency of PSII were reduced less in autotetraploid lines. PEPC activities were higher in autotetraploid rice lines. PEPC could alleviate inhibition of photosynthesis caused by drought stress. The chromosome-doubling enhanced rice photoinhibition tolerance under drought stress. The lower MDA content and superoxide anion production rate was found in the autotetraploid rice indicating low peroxidation level of cell membranes. At the same time, the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were higher in autotetraploid rice lines. SOD, POD, and CAT could effectively diminish the reactive oxygen species and reduced the membrane lipid peroxidation.     

6-苄（基）腺嘌呤和抗坏血酸对渗透胁迫时杨树光合作用光抑制的影响

研究了6-BA和A5A对渗透胁迫时杨树幼苗叶片光合作用光抑制和活性氧代谢的影响．结果表明,渗透胁迫时杨树叶片净光合速率(Pn)和表观量子效率(AQY)降低,光合作用光抑制加剧,超氧化物歧化酶(SOD)活性升高,抗坏血酸过氧化物酶(APX)活性降低,O2产生加快,H2O2和膜脂过氧化产物丙二醛(MDA)含量升高．6-BA和A5A预处理使胁迫时叶片SOD和APx活性升高。O2生成减少。H2O22和MDA含量降低,同时缓解了光合作用的光抑制．相关分析表明,杨树叶片活性氧水平和MDA含量与Pn和AQY呈负相关．胁迫时杨树叶片活性氧的积累与光合作用光抑制有一定关系,6-BA和A5A对光抑制的缓解作用与其对活性氧清除系统的促进作用有关。     

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.