Oxidative damage in unfertilized eggs of Chinese rare minnow (Gobiocypris rarus) exposed to nonylphenol

In the present study, female Chinese rare minnows (Gobiocypris rarus) were used as in vivo models and exposed to nonylphenol (NP) at concentrations of 1 to 200 mu g/L for 21 d under semistatic conditions. Molecular biomarkers of oxidative stress were measured in unfertilized eggs and included reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances [TBARS] and protein carbonyl), superoxide dismutase activity, and glutathione. Cathepsin D activity as an indicator of egg viability also was assayed. Nonylphenol induced ROS formation in unfertilized eggs in all exposed groups compared to the controls. The levels of protein carbonyl and TBARS in unfertilized eggs were significantly increased (p < 0.05) at 10 to 200 and 100 to 200 mu g/L, respectively. Good positive correlations were shown between ROS induction and levels of TBARS and protein carbonyl in eggs (R = 0.918, p < 0.05 and R = 0.784, p < 0.05, respectively). Superoxide dismutase activity in eggs was significantly inhibited (p < 0.05) in the 50 to 200 mu g/L exposure groups. Glutathione levels in eggs were significantly depleted (p < 0.05) at 100 to 200 mu g/L concentrations. In addition, ROS induction resulted in oxidative damage to lipid and protein in chorions. Significant reductions (p < 0.05) of the protein and lipid contents in chorions were both found in the 50 to 200 mu g/L exposure groups. A previous study found that NP exposure could lead to chorion thinning in zebra fish. Thus, the reductions in protein and lipid contents in chorion could be the reason for chorion thinning by NP exposure. Meanwhile, cathepsin D activity was significantly inhibited (p < 0.05) in all exposure groups. The results demonstrated that NP-induced oxidative stress could damage the chorion of unfertilized eggs and lead to a decline in gamete quality in female Chinese rare minnow.

Free radical scavenging and antioxidant enzymes activation of polysaccharide extract from Nostoc sphaeroides

Nostoc sphaeroides Kuetzing has been used as a traditional medicine in China to treat a variety of ailments. This research identified the antioxidant activities of polysaccharide extract from Nostoc sphaeroides. The extract, which contains 46.2% carbohydrates, exhibited an effective scavenging capability on superoxide radical, hydroxyl radicals in non site-specific as well as site-specific assays, and also performed lipid peroxidation inhibition in a dose-dependent manner. Polysaccharide extract had no 1,1-diphenyl-2-picrylhydrazyl radical scavenging potential at all test concentrations. Activities of superoxide dismutase, catalase, and glutathione peroxidase in human embryo kidney 293 cells were increased effectively when Nostoc sphaeroides extract was applied. These results suggested that the use of N. sphaeroides in treating ailments may be based on the antioxidant capacities of polysaccharide composition.

Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption

So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii, Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003-Sep. 2004 in Lake Taihu with toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the organs were done by LC-MS and HPLC. The major toxins were present in the hepatopancreas (45.5-55.4%), followed by visceral mass with substantial amount of gonad (27.6-35.5%), whereas gill and foot were the least (1.8-5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass, gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 mu g/g DW, respectively. There were rather good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r = 0.75-0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the hepatopancreas and the delta(13)C (r = 0.919) or delta(15)N (r = 0.878) of the foot, indicating that the different MC content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four bivalves were 8-23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk of consuming bivalves in Lake Taihu. (C) 2005 Wiley Periodicals, Inc.

Microcystin-RR-induced accumulation of reactive oxygen species and alteration of antioxidant systems in tobacco BY-2 cells

Microcystins are cyclic heptapeptide hepatoxins produced by cyanobacteria. It has been shown that microcystins have adverse effects on animals and on plants as well. Previous researches also indicated that microcystins were capable of inducing oxidative damage in animals both in vivo and in vitro. In this study, tobacco BY-2 suspension cell line was applied to examine the effects of microcystin-RR on plant cells. Cell viability and five biochemical parameters including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GPX) and peroxide dismutase (POD) were investigated when cells were exposed to 50 mg/L microcystin-RR. Results showed that microcystin-RR evoked decline of the cell viability to approximately 80% after treating for 144 h. ROS levels, POD and GPX activities of the treated cells were gradually increased with a time dependent manner. Changes of SOD and CAT activities were also detected in BY-2 cells. After 168 h recovery, ROS contents, POD, GPX and CAT activities returned to normal levels. These results suggest that the microcystin-RR can cause the increase of ROS contents in plant cells and these changes led to oxidant stress, at the same time, the plant cells would improve their antioxidant abilities to combat mirocystin-RR induced oxidative injury. (c) 2005 Elsevier Ltd. All rights reserved.

Determination of some reactive oxygen species scavengers based on the peroxidase-oxidase oscillator

This paper reports a new method for detection of ROS scavengers including superoxide dismutase, ascorbic acid and glutathione based on a 'probe' of peroxidase-oxidase biochemical oscillator. The oscillation period and amplitude change with different concentrations of scavengers. The linear ranges of superoxide dismutase, ascorbic acid and glutathione are respectively 1.56 x 10(-4)-1.56 x 10(-3) mg mL(-1), 1.75 x 10(-7) -1.75 x 10(-5) mol L-1 and 9.38 x 10(-7) -7.5 x 10(-5) mol L-1. The selectivity, linearity and precision for superoxide dismutase, ascorbic acid, and glutathione are presented and discussed. The results compared well with other standard methods for determination of superoxide dismutase, ascorbic acid and glutathione. Some possible steps in the overall reaction mechanisms are discussed.

Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae)

The genome segments 1, 2, and 3 of the grass carp reovirus (GCRV), a tentative species assigned to genus Aquareouirus, family Reouiridae, were sequenced. The respective segments 1, 2, and 3 were 3949, 3877, and 3702 nucleotides long. Conserved moths 5' (GUUAUUU) and 3' (UUCAUC) were found at the ends of each segment. Each segment contains a single ORF and the negative strand does not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VPI might represent the viral guanyly/methyl transferase (involved in the capping process of RNA transcripts) and VP3 the NTPase/helicase (involved in the transcription and capping of viral RNAs), The highest amino acid identities (26-41%) were found with orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses, and orthoreoviruses, It should also permit to precise the taxonomic status of these different viruses. (C) 2000 Academic Press.

人源抗体酶GPX的可溶性表达及生物活性研究

谷胱甘肽过氧化物酶 (Glutathione Peroxidase, GPX)，是生物体内抗氧化应激酶系的重要成员，它可以清除脂质氢过氧化物（ROOH）及过氧化氢 (H_2O_2)，保护机体免受活性氧的损伤或减少活性氧对机体的损伤程度。为了解决鼠源抗体酶GPX应用于人体时会诱发人抗鼠反应 (Human Anti-Mouse Antibody)这一问题，我们实验室从人单链抗体库中筛选了特异的抗体，经化学修饰后得到了具有GPX活性的抗体酶。本文在此工作的基础上，对己筛选出的人单链抗体基因进行了可溶性表达，并用化学修饰的方法进行硒化，得到了GPX活力为80U/μmol的抗体酶。根据优化的表达条件，直接得到了具有天然结构的抗体蛋白，省去了原来的包含体表达中的变性、复性步骤。且所得的抗体为融合蛋白，在蛋白的末端接有6个连续的组氨酸，因此用Ni-NTA亲和层析柱对其进行了一步纯化，即得到了纯抗体蛋白。从而大大的简化了抗体酶的制备过程。本论文还对抗体酶的生物活性进行了研究。采用H_2O_2/Fe~(2+) 自由基发生系统，分别以线粒体的膨胀度、乙二醛生成量和自由基含量为不同的损伤指标进行测量，研究了抗体酶对自由基损伤的牛心线粒体的保护作用。实验结果表明，硒化后的抗体酶具有较强的保护作用。

荞麦(Fagopyrum tataricum Gaertn.和F. esculentum Moench.)对紫外线B辐射的响应研究

近二十多年来，基于对臭氧层衰减、紫外线B（UV-B）增强的担心，研究者希望了解到紫外线辐射对不同作物的影响情况，增强UV-B辐射条件下是否对作物的生长发育、产量质量构成威胁。在本试验中，我们首先探讨了双子叶作物黄瓜（Cucumis sativus）和大豆（Glycine max）对不同紫外波段的生物效应[分别为B-UVA（315-400 nm），N-UVA（315-340 nm），B-UVB（275-400 nm）和N-UVB（290-340 nm），UV-（>400nm）作对照]。我们观察到所有的UV波段处理都使黄瓜和大豆的生长受到抑制，并且细胞受到不同程度的氧化伤害；UV波段处理的作用效果与不同波段的紫外有效生物辐射剂量有关。处理差异在UV-B波段内部和UV-A波段内部同样存在。植物生长UV辐射公式（BSWF）能很好的预测本试验UV-B波段内的平均植物效应，但不能预测UV-A波段的植物效应。短波UV-A的生物作用强于长波UV-A。光合色素的变化与UV波谱差异和种间差异有关。在高的紫外/可见光背景下，UV-A处理同UV-B同样导致光合色素的降低，但黄瓜类胡萝卜素/叶绿素比例升高。与其他研究者的试验结果比较后，我们认为紫外线B辐射的生物效应一致性很高，但紫外线A波段的生物学效应存在较大争议。因此我们在本试验的基础上仅进行荞麦[苦荞(Fagopyrum tataricum Gaertn.)和甜荞(Fagopyrum esculentum Moench.)]对紫外线B波段的响应研究。 我们对苦荞品种-圆籽荞进行了连续两个生长季节的大田半控制试验以观察UV-B辐射对苦荞生长、发育、产量及叶片色素的影响；试验小区进行降低UV-B、近充足UV-B和增强UV-B辐射处理。我们的试验表明在不同强度UV-B辐射下苦荞的生长、地上部生物量积累及最终产量都有所下降，但苦荞的发育加快；当前条件下的日光紫外线B辐射对植物生长和产量也造成负面影响。植物光合色素被日光及增强UV-B辐射降低；UV化合物及卢丁含量在中低剂量的UV-B辐射强度下显著升高，但在高剂量的增强UV-B辐射下短期升高后迅速下降。我们的试验表明苦荞是一个对UV-B高度敏感的作物。苦荞对UV-B的敏感性与UV-B剂量、外界环境因素及生长季节有关。 单个苦荞品种的试验结果使我们认识到外界UV-B辐射已经对苦荞生长发育构成逆境条件，未来全球气候变化条件下增强紫外线B辐射可能使其处于更不利的生长环境中。因此我们有进行了多个种群进行UV-B响应观察并筛选耐性种群。我们对15个苦荞种群进行增强UV-B辐射处理（6.30 kJ m2 UV-BBE，模拟当地25%的臭氧衰减），我们观察苦荞UV-B辐射效应存在显著的种内差异，UV-B辐射对多数种群具有抑制作用，但对一些种群还有刺激作用。我们采用主成分分析方法与作物UV-B响应指数（RI）来评价苦荞作物UV-B辐射耐性。我们发现作物的UV-B耐性不仅与其原产地背景UV-B强度有关，而且与作物相对生长效率、次生代谢产物含量（如卢丁）及其他因素有关。我们观察到苦荞伸展叶总叶绿素变化与UV-B耐性成正相关；室内苦荞幼苗的UV-B辐射致死试验表明：苦荞种群死亡率与其UV-B耐性成负相关。 此外，我们对甜荞的UV-B辐射响应也进行了初步研究。选取美姑甜荞、巧家甜荞和云龙甜荞进行5个梯度的增强UV-B辐射室外模拟试验。我们观察到UV-B辐射显著降低了甜荞的生长、生物量及产量；并严重影响了甜荞的生殖生长，降低了花序数、种子数和结实率；并且UV-B辐射对甜荞的抑制作用存在显著的剂量效应。三种甜荞品种存在显著的种内差异，其中美姑品种UV-B耐性最强，且膜脂受UV-B辐射氧化伤害最小，这与该品种UV-B辐射下较高的GR酶活性、APX酶活性和PPO酶活性、以及含量更高的抗坏血酸有关。甜荞的次生代谢也受到增强UV-B辐射的影响，其香豆酰类化合物在UV-B辐射下升高显著，而槲皮素含量也在高剂量UV-B辐射下有所增加；卢丁含量依赖UV-B辐射剂量而变化，中低剂量UV-B辐射下其卢丁含量逐渐升高，但在高剂量辐射下逐渐下降。 通过对生长在高海拔地区的荞麦作物(苦荞和甜荞)进行的室外研究，我们认识到作物不同品种存在很大的耐性差异，这就为UV-B耐性育种创造了有利条件。进一步加大荞麦种质资源筛选力度并深入认识荞麦抗性机理，在此基础上通过杂交或其他基因融合手段培育抗性品种，对高剂量UV-B辐射地区的荞麦产量的提高将起到重要推动作用，并使荞麦生产能有效应对未来全球气候变化条件下UV-B辐射可能升高的威胁。 During last few decades, due to concern of ozone layer depletion and enhancement of ultraviolet B radiation（UV-B, 280-315 nm), the agronomist want to know the responses of different crop species to UV-B. In the first experiment of our study, the effect of different UV band [B-UVA（315-400 nm), N-UVA（315-340 nm), B-UVB（275-400 nm), N-UVB（290-340 nm）and UV-（>400nm, as control)] on the cucumber（Cucumis sativus）and soybean（Glycine max）were investigated in growth room. Spectra-dependent differences in growth and oxidation indices existed within UV-A bands as well as UV-B bands. The general biological effects of different band were UV- ＜ B-UVA＜ N-UVA＜N-UVB＜B-UVB. The plant growth biologically spectra weighting function（BSWF）matched well with average plant response in UV-B region, but not in UV-A region. Shorter UV-A wavelength imposed more negative impact than longer UV-A wavelength did in both species. The effect on photosynthetic pigment was related to different UV bands and different species. The photosynthetic pigment content was decreased by UV-A spectra as well as UV-B spectra. In comparison with the results of previous studies, we found that the wavelength-dependent biological effect of ultraviolet B radiation has high consistency, but the biological effect of ultraviolet-A radiation was inconsistent. We narrow our following study on the effect of ultraviolet B radiation on the buckwheat（tartary buckwheat and common buckwheat). The tartary buckwheat（Fagopyrum tataricum Gaertn.）cultivars Yuanziqiao was grown in the sheltered field plots for two consecutive seasons under reduced, near-ambient and two supplemental levels of UV-B radiation. The crop growth, photosynthetic pigments, total biomass, final seed yield and thousand-grain weight were decreased by near-ambient and enhanced UV-B radiation, while crop development was promoted by enhanced UV-B radiation. Leaf rutin concentration and UV-B absorbing compound was generally increased by UV-B with the exception of 8.50 kJ m-2 day-1 supplemental levels. Our results showed that tartary buckwheat is a potentially UV-B sensitive species. Study on one cultivars showed that ambient solar radiation had present a stress to tartary buckwheat. This makes it necessary to observe the UV-B response of many cultivars and screen tolerant cultivars. Fifteen populations of tartary buckwheat were experienced enhanced UV-B radiation simulating 25% depletion of the stratospheric ozone layer in Kunming region, and plant responses in growth, morphology and productivity were observed. Principal components analysis（PCA）was used to evaluate overall sensitivity of plant response to UV-B as well as response index. The different populations exhibited significant differences in responses to UV-B. The photosynthetic pigments of young seedlings were also affected significantly under field condition. On the other hand, the healthy seedlings of different populations were exposed to the high level of UV-B radiation in growth chambers to determine the plant lethality rate. The plant tolerance evaluated by multivariate analysis was positively related to total plant chlorophyll change, but negatively related to lethality rate. In other hand, the UV-B responses of the other important cultivated buckwheat species, common buckwheat（Fagopyrum esculentum Moench.), were also studied preliminarily. Three widespread cultivated variety（Meigu, Qiaojia and Yunlong cultivars）were provided with five level of enhanced UV-B radiation outdoors. We observed that the crop growth, development and production were significantly decreased, and reproductive production, like anthotaxy number, seed number and seed setting ratio, was also decreased. Dose-dependent inhibition effect caused by enhanced UV-B radiation also existed in common buckwheat. Significant intraspecific difference existed in those three cultivars. The Meigu cultivars with dwarfed growth and lower production have highest UV-B tolerance as well as lowest damage in cell membrane, this could be associated with profound enhancements of glutathione reductase（GR）activity, ascorbate peroxidase activity and polyphenol oxidase activity as well as higher ascorbic acid concentration. The secondary metabolism was also affected by UV-B radiation, with profound elevation of coumarin compound and moderate increase of quercetin concentration. Rutin concentration was peaked in 5kJ m-2 UV-B. The contrasting effect of UV-B radiation on different populations indicated that there existed abundant genetic resources for selecting tolerant populations of common and tartary buckwheat. Much effort needed be pose on screening of buckwheat germplasm and clarification of mechanism of buckwheat tolerance to UV-B. On this base the tolerant cultivars could be bred by hybridization and other gene transfusion method, this would help increase buckwheat yield in high ambient UV-B region and counteract the effect of possible enhanced UV-B radiation in future.

青杨组(Section Tacamahaca Spach)杨树不同种群对土壤干旱和重金属锰污染的生态生理响应

土壤是人类赖以生存的自然环境和农业生产的重要资源，世界面临的粮食、资源和环境问题与土壤密切相关，目前危害土壤的主要因素是干旱和重金属污染。杨树具有适应性强、生长快和丰产等特性，本论文以青杨组杨树为模式植物，采用植物生态、生理及生物化学等方法，研究杨树对土壤干旱和锰胁迫的生态生理反应以及种群间差异，研究成果可为我国干旱半干旱地区营造人工林、防止沙漠化提供理论依据，也为恢复与重建重金属污染地区退化生态系统提供科学指导。主要研究结果如下： 1. 青海杨不同种群对干旱胁迫的响应差异 干旱胁迫显著降低了两个青海杨种群（干旱种群和湿润种群）生物量积累，包括株高、基径、干物质积累等，通过植物结构的调整，有更多的生物量向根部分配。干旱胁迫还显著降低了叶绿素和类胡萝卜素含量，增加了游离脯氨酸和总氨基酸含量。另一方面，干旱胁迫诱导了活性氧的积累，作为第二信使，激活了抗氧化系统，包括抗坏血酸（ASA）含量和酶系统如超氧化物歧化酶（SOD），愈创木酚过氧化物酶（GPX），抗坏血酸过氧化物酶（APX）和谷胱甘肽还原酶（GR）。这样，杨树既有避旱机制又有耐旱机制，使其在干旱胁迫下有相当程度的可塑性。与湿润种群相比，干旱种群杨树有更多的生物量分配到根部，积累了更多的游离脯氨酸和总氨基酸来进行渗透调节，并且有更有效的抗氧化系统，包括更高含量的ASA 和更高活性的APX 和GR，这些使得干旱种群杨树比湿润种群杨树对干旱有更好的耐性。 2. 喷施硝普钠（SNP）对青海杨阿坝种群干旱胁迫耐性的影响 干旱胁迫显著的降低了青海杨阿坝种群的生长和生物量积累以及叶片相对含水量，还诱导了脯氨酸的合成以进行渗透调节。干旱胁迫下过氧化氢（H2O2）显著累积从而造成对膜脂和蛋白的伤害，使得丙二醛和蛋白羰基含量升高。干旱胁迫下喷施SNP可以减轻干旱胁迫造成的伤害，包括增加叶片的相对含水量，增加脯氨酸和总氨基酸的积累，并激活抗氧化酶系统如SOD，GPX和APX，从而减少丙二醛（MDA）和蛋白羰基（C＝O）的积累，但是在水分良好情况下SNP的效果不显著。 3. 青杨不同种群对锰胁迫的生长与形态响应差异 在同一锰浓度下，干旱种群的耐性指数都要高于湿润种群，这表明青杨对干旱和高锰胁迫具有交叉耐性。两个种群的株高，生物量和叶绿素含量都随锰浓度的升高而逐渐下降。就累积浓度而言，0 和0.1 mM 锰胁迫下，干旱种群积累的锰浓度要高于湿润种群，而在高浓度锰胁迫下(0.5 和1 mM)，湿润种群要高于干旱种群。在0，0.1 和0.5 mM下，锰大多积累在根中，叶片次之，茎中最少。而在1 mM，锰更多的积累在叶片中。就累积总量而言，在各个锰浓度胁迫下，根，茎和叶相比，两个种群青杨都是叶片累积的锰总量要高于根和茎。两个种群间比较，对照中没有显著区别，0.1 mM 锰胁迫下，湿润种群根中累积的锰要高于干旱种群，而在地上部中，干旱种群要高于湿润种群。而0.5 和1 mM 锰胁迫下，根、叶、茎+叶、根+茎+叶中，锰累积总量都是湿润种群高于干旱种群。锰胁迫下，青杨叶片数和叶面积包括总叶面积和平均叶面积都显著降低。叶片横切面的光学显微观察结果表明未经锰胁迫的栅栏组织的细胞饱满，海绵组织发达、清晰；胁迫后杨树叶片栅栏组织细胞出现不同程度的皱缩，海绵组织几乎不可见，此外还发现输导组织在胁迫下密度变小和分生组织严重割裂等现象。 4. 青杨不同种群对锰胁迫的生理与生化响应差异 青杨两个种群脱落酸（ABA）含量在锰胁迫下都显著增加，干旱种群的增幅更大。三种多胺含量在锰胁迫下显示了不同的响应趋势：腐胺在两个种群的各个锰处理下都增加，亚精胺只在干旱种群中显著增加，而精胺除了干旱种群在1 mM 下有所增加外，在锰胁迫下变化很小。谷胱甘肽含量随锰浓度升高而增加，在0.5 mM 锰时达到最高值，1mM 时有所下降。植物络合素（PCs）含量与非蛋白巯基（NP-SH）趋势相似，随锰浓度的升高而增加，且干旱种群中含量要高于湿润种群。锰处理还引起氧化胁迫，表现为过氧化氢和丙二醛含量增加。SOD 活性在湿润种群中，在0 到0.5 mM 锰胁迫下活性升高，但在1 mM 锰胁迫时，其活性有所下降。而在干旱种群中，SOD 活性变化较小，并始终维持在一个较高的水平。APX 活性在两个种群中都随锰浓度的升高而增加，干旱种群活性要高于湿润种群。锰胁迫还显著增加了酚类物质的含量，同时GPX 和多酚氧化酶（PPO）活性也随锰浓度的升高而增加。干旱种群的酚类含量和GPX 与PPO 活性都要高于湿润种群。锰胁迫还改变了氨基酸的含量和构成，根据锰胁迫下浓度变化的不同，可以将游离氨基酸分为三组：第一组包括，谷氨酸，丙氨酸和天门冬氨酸，这一组氨基酸含量在锰胁迫下有所下降。第二组包括缬氨酸，亮氨酸和苏氨酸含量在锰胁迫下基本不变化或变化很小。剩下的氨基酸为第三组，这组氨基酸含量在锰胁迫下显著增加，而根据增加的幅度又可以将它们分为两个亚组，丝氨酸，酪氨酸，苯丙氨酸，组氨酸和脯氨酸，在1 mM 下的含量是对照的4 倍以上。异亮氨酸，赖氨酸，精氨酸和甘氨酸含量在1 mM 下是对照含量的2 倍以下。同时，同一锰浓度下，干旱种群比湿润种群积累的氨基酸含量要高。 Soil is the indispensable environment for human survival and important resource foragriculture development. Food and environmental problems facing the world are all closelyrelated to soil and nowadays it is threatened by many factors, among which drought stress andheavy metal pollution are the most serious ones. Poplars (Populus spp.) are importantcomponents of ecosystem and suitable as a source of fuel, fiber and lumber due to their fastgrowth. In this study, different populations of Section Tacamahaca spach were used as modelplants to investigate the adaptability to drought stress and manganese toxicity and differencesbetween populations from dry and wet climate regions. Our results can provide theoreticalevidence for the afforestation and prevention of desertification in the arid and semi-arid areas,and also can supply scientific direction for the reconstruction and rehalibitation of ecosystemscontaminated by heavy metals. The results are as follows: 1. Differences in ecophysiological responses to drought stress in two contrastingpopulations of Populus przewalskii Drought stress not only significantly affected dry mass accumulation and allocation, butalso significantly decreased chlorophyll pigment contents and accumulated free proline andtotal amino acids. On the other hand, drought also significantly increased the levels ofabscisic acid and reactive oxygen species, as secondary messengers, to induce the entire set ofantioxidative systems including the increase of reduced ascorbic acid content and the activities of superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and glutathioneredutase. Thus the combination of drought avoidance and tolerance mechanisms conferredpoplar a high degree of plasticity in response to drought stress. Compared with the wetclimate population, the dry climate population showed lower dry matter accumulation andallocated more biomass to root systems, and accumulated more free proline and total aminoacids for osmotic adjustment. The dry climate population also showed more efficientantioxidant systems with higher content of ascorbic acid and higher activities of ascorbateperoxidase and glutathione redutase than the wet climate population. All these made the dryclimate population superior in adaptation to drought stress than the wet climate population. 2. Effect of exogenous applied SNP on drought tolerance in Populus przewalskii Drought stress significantly increased hydrogen peroxide content and caused oxidativestress to lipids and proteins assessed by the increase in malondialdehyde and total carbonylcontents, respectively. The cuttings of P. przewalskii accumulated proline and other aminoacids for osmotic adjustment to lower water potential, and activated the antioxidant enzymes such as superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase to maintain thebalance of generation and quenching of reactive oxygen species. Moreover, exogenous SNPapplication significantly heightened the growth performance of P. przewalskii cuttings underdrought treatment by promotion of proline accumulation and activation of antioxidant enzymeactivities, while under well-watered treatment the effect of SNP application was very little. 3. Morphological responses to manganese toxicity in the two contrasting populations ofPopulus cathayana High concentration of manganese caused significant decrease in shoot height andbiomass accumulation. The tolerance index of the dry climate population was significantlyhigher than that of the wet climate population, suggesting the superior Mn tolerance in theformer and the existence of cross-tolerance of drought stress and high Mn toxicity. Injuries tothe leaf anatomical features were also found as the reduced thickness in palisade and spongyparenchyma, the decreased density in the conducting tissue and the collapse and split in themeristematic tissue in the central vein. As for the Mn concentrations in the plant tissues, under0, 0.1 and 0.5 mM, most of the Mn accumulated in the roots, then leaves, and stem the least, while under 1 mM, most of the Mn accumulated in the leaves. As far as the total amounts ofMn extraction are concerned, the leaf extracted more Mn than the root and stem in the twopopulations under various Mn concentrations. There is no difference between the twopopulations under control. Under 0.1 mM, the wet climate population extracted higher Mn inthe root than the dry climate population, while in the shoot, the dry climate populationextracted much more Mn. Under 0.5 and 1 mM, the wet climate population translocated moreMn both in the root and the shoot than the dry climate population. 4. Physiological and biochemical responses to manganese toxicity in the two contrastingpopulations of Populus cathayana Mn treatment resulted in oxidative stress indicated by the oxidation to lipids, proteinsand DNA. A regulated network of defence strategies was employed for the chelation,detoxification and tolerance of Mn including the enhanced synthesis of ABA and polyamines,the accumulation of free amino acids, especially His and Pro, and the activation of theenzymes superoxide dismutase and guaiacol peroxidase. Contents of non-protein thiol,reduced glutathione, phytochelatins and phenolics compounds and activities of superoxide dismutase, guaiacol peroxidase and polyphenol oxidase also increased significantly forantioxidant or chelation functions. The wet climate population not only accumulated lessabscisic acid, free amino acids, phytochelatins and phenolics compounds, but also exhibitedlower activities of superoxide dismutase, guaiacol peroxidase and polyphenol oxidase thusresulting in more serious oxidative damage and more curtained growth.

中国沙棘(Hippophae rhamnoides subsp. sinensis)不同种群对干旱胁迫的响应差异与干旱诱导蛋白的分析

沙棘广泛分布于亚欧大陆的温带地区和亚洲亚热带的高海拔地区。沙棘能适应多种生态环境，能耐受多种逆境(如干旱、低温、高温和盐害等)。在中国，沙棘常常被用作植被恢复中的先锋树种而大量栽培。本文以中国沙棘为试验材料，探索沙棘适应干旱机制，以及沙棘对干旱胁迫的适应机制是否存在种群间的差异，同时试图通过分析干旱胁迫下沙棘叶片蛋白质表达变化探索沙棘适应干旱胁迫的分子机理。 对三个分别来自低海拔湿润地区、低海拔干旱地区和高海拔湿润地区的中国沙棘种群进行干旱胁迫处理。干旱胁迫能提高根冠比，比叶面积，降低平均叶面积和总生物量，提高沙棘的抗氧化性酶活性、脯氨酸含量、脱落酸(ABA)含量、降低光合作用，提高长期用水效率。实验中的这两个低海拔种群比高海拔种群抵抗干旱的能力更强,不同的种群采用了不同的策略来耐受干旱胁迫和过氧化胁迫。 在2004 年度的实验中，干旱胁迫处理下，高海拔湿润种群(道孚种群)严重失水，生长也受到更大的抑制，非气孔因素在抑制光合作用方面占支配地位，抗坏血酸含量下降，ABA和脯氨酸含量增加幅度比九寨沟种群的要高，这可能是因为道孚种群严重失水而引起的；而低海拔湿润种群(九寨沟种群)的体内水分状况几乎不受干旱的影响，生长情况也较道孚种群要好。 在2005 年度的试验中，和高海拔湿润地区种群(道孚)相比较，低海拔干旱地区种群(定西)在叶片相对水含量、根冠比、抗氧化酶活性(过氧化氢酶、抗坏血酸过氧化物酶和谷胱甘肽过氧化物酶)、保护性物质(脯氨酸，脱落酸)含量等方面都要高，光能热耗散能力也更强，而且气体交换参数(气孔扩散阻力和胞间CO2浓度等)对干旱也更不敏感。 分析了干旱胁迫下沙棘叶片蛋白质表达的变化。共发现319 个蛋白质，有4 个蛋白在干旱胁迫下消失(Putative ABCtransporter ATP-binding protein 、Hypothetical proteinXP-515578，热激蛋白Hslu219 和一个没得到鉴定的蛋白)，4 个只在干旱胁迫下出现(没命名的蛋白质产物，对甲基苯-丙酮酸双加氧酶，NTrX 和一个没得到鉴定的蛋白)，46 个蛋白质的表达丰度变化显著，包括32 个干旱负调蛋白，14 个干旱正调蛋白(3 个Rubisco 的大亚基、J-type–co-chaperone Hsc20、putative protein DSM3645-2335、putative acyl-COA 脱氢酶、nesprin-2 和两个没有得到鉴定的蛋白质)。这些蛋白质参与了氮代谢调控、抗氧化行物质的合成、脂肪酸β－氧化、核骨架构造、[Fe-S]基团组装、物质跨膜运输、细胞分裂或作为分子伴侣和蛋白质酶起作用。putative ABC transporter ATP-binging protein、NtrX、nesprin-2 和Hslu 是本试验新发现的高等植物蛋白，我们主要从它们的保守结构域或在其他生物中的同源物来猜测它们的功能。实验结果为我们研究植物抗干旱机制提供了新线索和新视野。 Seabuckthorn (Hippophae rhamnoides L.) is widly distributed throughtout the temperatureresiogn of Europe and Asia and sub-tropical plateau zone of Asia. H. rhamnoides can adapatseveral different environments, and can tolerant several envioronmental stresses (e.g, lowtemperature, high temperature, drought and salty). It has been widely used in forest restoration asthe pioneer species in China. In present study, we applied H.rhamnoides subsp. Sinensis asexperimental materials to study its drought-tolerant mechanism, and expected to findpopulational difference in drought-tolerant mechanism that may exist among populations, and tryto get some insight in drought-tolerant mechanism of it at morecular level through analyzing thechange of leaf protein expression. Three populations from high altitude wet zone, low altitude wet zone and low altitude arid znoe,respectively, were applied in our experiment, and were subjected to drought. Drought increasedthe root/shoot ratio(RS), special leaf area, long-term water use efficinency, activity of antioxidantenzymes, proline content and abscisic acid (ABA) content, declined the net photosynthesis rate(A), average leaf area (ALA), total biomass (TB). Both two low altitude populations were moredrought-tolerant than the high altitude population, and different population applied differentstratedgies to tolerant oxidant stress and drought stress. The results of the exprement in 2004 showed that Daofu population was more drought-sensitivethan Jiuzhai population. Under drought conditions, leaf relative water content (RWC) greatlydecreased in Daofu population, but not in Jiuzhai population. The large loss of water in Daofupopulation resulted in a limitation on A mainly caused by non-stomatal factors, severer suppression in growth rate and a significant reduction in ascorbic acid (AsA) content, comparedwith Jiuzhai population. The greater increase in content of ABA and proline in Daofu populationmay be also induced by large loss in water, so that enable plants to cope with sever drought. In the exprement of 2005, drought significantly increased RS, activities of catalase (CAT),peroxidase (POD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX), and alsosignificantly increased ABA and proline contents. On the other hand, compared with Daofupopulation, drought induced larger RS and activities of CAT, GPX and APX, and higher ABAcontent in Dingxi population, whereas gas exchange traits, e.g., stomatal limitation value (LS) andintercellular CO2 concentration (Ci), were less responsive to drought in Dingxi population thanthose in Daofu population. All these factors enable Dingxi population to tolerant drought betterthan Daofu population. The leaf protein profile of seabuchthorn subjected to drought was analyzed. Altogether 319proteins were detected in well-watered sample, four proteins disappeard by drought (putativeABCtransporter ATP-binding protein, hypothetical protein XP-515578, Hslu219and aunidentified protein), four only appeared under drought (a probable nitrogen regulation protein(NtrX), a 4-hydroxyphenylpyruvate dioxygenase , an unnamed protein product and an identified protein), 32 drought down-regulated proteins, and 14 drought up-regulated proteins (nine wereidentified: three large subunits of Rubisco, a hypothetical protein DSM3645-23351, a putativeacyl-COA dehydrogenase, a nesprin-2, a J-type-co-chaperone HSC20 and two unmatchedproteins). These proteins may involve in β-oxidation, cross-membrane transport, cell division,cytoskeleton stabilization, iron-sulfur cluster assembly, nitrogen metabolism regulation andantioxidant substance biosynthesis or function as molecular chaperone or protease. Four proteins(a putative ABC transporter ATP-binging protein, NtrX, nesprin-2, Hslu) were new found in highplants, and their functions were estimated from their conserved domain or their homologues inother organism. Our results provided new clue and new insight for us to study thedrought-tolerant mechanism in plants.

N-乙酰半胱氨酸对碳离子照射小鼠急性损伤的保护效应

探讨了N-乙酰半胱氨酸(N-acetylcysteine,NAC)对12C6+离子照射小鼠损伤的保护效应及可能的作用机制。预先给予昆明小鼠NAC(200mg/kg),后进行12C6+离子束4Gy的单次全身照射。照射后2h处死小鼠,取肝、肺组织,用化学法检测组织中超氧化物岐化酶(Superoxide dismutase,SOD)的活性,谷胱甘肽(Glutathione,GSH)含量及脂质过氧化产物丙二醛(Malondialdehyde,MDA)水平;用碱性单细胞凝胶电泳法检测DNA单链断裂;用流式细胞术检测组织细胞凋亡率。与照射对照组相比,提前给予NAC可极显著地减轻12C6+离子导致的肝、肺组织中DNA断裂(p<0.001)和细胞凋亡(p<0.001),并显著地提高组织中GSH的含量;NAC还明显地抑制了肺组织中碳离子辐照所致的MDA水平增高(p<0.01)并显著地诱导了组织中SOD活性的升高(p<0.01)。提示NAC可通过抵御组织内的氧化作用,合成、补充GSH的含量,阻止DNA链的断裂和细胞的凋亡,实现对碳离子辐照损伤的保护效应。

碳离子全身辐照对小鼠血清及肝脏中超氧化物歧化酶活性、丙二醛含量和脑组织中还原性谷胱甘肽浓度的影响

用12C6+离子束对小鼠进行吸收剂量分别为0.05、0.1、0.3、0.5、0.75、1、1.5、2Gy的一次性全身照射,5d后测定血清及肝脏中超氧化物歧化酶(Superoxide dismutase,SOD)活性和丙二醛(Maleicdialdehyde,MDA)含量,以及脑组织中还原性谷胱甘肽(Glutathione,GSH)的浓度。结果显示,吸收剂量小于0.75Gy小鼠的血清及肝脏中SOD活性高于对照组,大于0.75Gy则低于对照组;吸收剂量小于0.3Gy小鼠的血清及肝脏中MDA含量小于对照组,大于0.3Gy则大于对照组;吸收剂量小于0.5Gy小鼠的脑组织GSH浓度大于对照组,大于0.5Gy则小于对照组。低剂量的重离子全身辐照对小鼠的抗氧化系统有一定的激活作用,随着照射剂量的增大,抗氧化酶活性明显降低,脂质过氧化水平增高。

Effects of Naotan Pill on repair of neural cells and cognitive disorders in juvenile rats following hypoxia and ischemia

BACKGROUND: Hypoxia and ischemia induce neuronal damage, decreased neuronal numbers and synaptophysin levels, and deficits in learning and memory functions. Previous studies have shown that lycium barbarum polysaccharide, the most effective component of barbary wolfberry fruit, has protective effects on neural cells in hypoxia-ischemia. OBJECTIVE: To investigate the effects of Naotan Pill on glutamate-treated neural cells and on cognitive function in juvenile rats following hypoxia-ischemia. DESIGN, TIME AND SETTING: The randomized, controlled, in vivo study was performed at the Cell Laboratory of Lanzhou University, Lanzhou Institute of Modern Physics of Chinese Academy of Sciences, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from December 2005 to August 2006. The cellular neurobiology, in vitro experiment was conducted at the Institute of Human Anatomy, Histology, Embryology and Neuroscience, School of Basic Medical Sciences, Lanzhou University, and Department of Traditional Chinese Medicine of Gansu Provincial Rehabilitation Center Hospital, China from March 2007 to January 2008. MATERIALS: Naotan Pill, composed of barbary wolfberry fruit, danshen root, grassleaf sweetflag rhizome, and glossy privet fruit, was prepared by Gansu Provincial Rehabilitation Center, China. Rabbit anti-synaptophysin, choline acetyl transferase polyclonal antibody, streptavidin-biotin complex kit and diaminobenzidine kit (Boster, Wuhan, China), as well as glutamate (Hualian, Shanghai, China) were used in this study. METHODS: Cortical neural cells were isolated from neonatal Wistar rats. Neural cell damage models were induced using glutamate, and administered Naotan Pill prior to and following damage. A total of 54 juvenile Wistar rats were equally and randomly assigned into model, Naotan Pill, and sham operation groups. The left common carotid artery was ligated, and then rat models of hypoxic-ischemic injury were assigned to the model and Naotan Pill groups. At 2 days following model induction, rats in the Naotan Pill group were administered Naotan Pill suspension for 21 days. In the model and sham operation groups, rats received an equal volume of saline. MAIN OUTCOME MEASURES: Neural cell morphology was observed using an inverted phase contrast microscope. Survival rate of neural cells was measured by MTT assay. Synaptophysin and choline acetyl transferase expression was observed in the hippocampal CA1 region of juvenile rats using immunohistochemistry. Cognitive function was tested by the Morris water maze. RESULTS: Pathological changes were detected in glutamate-treated neural cells. Neural cell morphology remained normal after Naotan Pill intervention. Absorbance and survival rate of neural cells were significantly greater following Naotan Pill intervention, compared to glutamate-treated neural cells (P < 0.05). Synaptophysin and choline acetyl transferase expression was lowest in the hippocampal CA1 region in the model group and highest in the sham operation group. Significant differences among groups were observed (P < 0.05). Escape latency and swimming distance were significantly longer in the model group compared to the Naotan Pill group (P < 0.05). CONCLUSION: Naotan Pill exhibited protective and repair effects on glutamate-treated neural cells. Naotan Pill upregulated synaptophysin and choline acetyl transferase expression in the hippocampus and improved cognitive function in rats following hypoxia-ischemia.

Antioxidant N-acetylcysteine attenuates the acute liver injury caused by X-ray in mice

The aim of this study was to evaluate the protective effects of different doses and administration modes of N-acetylcysteine (NAC) against X-ray-induced liver damage in mice. Kun-Ming mice were divided into four groups, each composed of six animals: two control groups and two NAC-treated groups. An acute study was carried out to determine alterations in lipid peroxidation (determined by measuring malondiadehyde (MDA) level), glutathione (GSH) content and superoxide dismutase (SOD) activity (assayed by colorimetric method), and DNA damage (characterized by DNA-single strand break using with comet assay) as well as cell apoptosis (measured by flow cytometry) at 12 h after irradiation. The results showed that there were dose-related decreases in MDA level, DNA damage and cell apoptosis, and dose-dependent increases in GSH content and SOD activity in all NAC-treated groups compared to control groups, indicating that pre-treatment or post-treatment with NAC significantly attenuates the acute liver damage caused by X-ray. In addition, significant positive correlations were observed between MDA level and DNA damage or cell apoptosis, implying that lipid peroxidation plays a major role in X-ray-induced liver injury. The data suggest that NAC exerts its radioprotective effect by counteracting accumulated reactive oxygen species in the liver through its properties as a direct antioxidant and a GSH precursor, when administered before or after X-ray irradiation.

Radio-resistance induced by nitric oxide to heavy ion irradiation in A172 human glioma cells

To investigate effects of nitric oxide on cellular radio-sensitivity, three human glioma cell lines, i.e. A172, A172 transfected green fluorescence protein (EGFP) gene (EA172) and A172 transfected inducible nitric oxide synthesis (iNOS) gene (iA72), were irradiated by C-12(6+) ions to 0, 1 or My. Productions of nitric oxide and glutathione (GSH) in A172, EA172 and iA172 were determined by chemical methods, cell cycle was analyzed by flow cytometry at the 24th hour after irradiation, and survival fraction of the cells was measured by colorimetric MTT assay at the 5th day after irradiation. The results showed that the concentrations of nitric oxide and GSH in iA172 were significantly higher than in A172 and EA172; the G(2)/M stage arrest induced by the C-12(6+) ion irradiation was observed in A172 and EA172 but not in iA172 at the 24th hour after exposure; and the survival fraction of iA172 was higher than that of EA172 and iA172. Data suggest that the radio-sensitivity of the A172 was reduced after the iNOS gene transfection. The increase of GSH production and the change of cellular signals such as the cell cycle control induced by nitric oxide may be involved in this radio-resistance.