测定血中亚硝酸盐的荧光分光光度法

目的:建立检测血中亚硝酸盐的荧光分光光度法.方法:采用硫酸-磷钨酸去蛋白预处理的方法排除血红蛋白等对测定的干扰,以2,3-二氨基萘与亚硝酸盐反应生成荧光化合物,用荧光分光光度法测定.结果:血标本以硫酸-磷钨酸去蛋白预处理2次,2,3-二氨基萘浓度为0.63 mmol·L-1,反应液和终止反应后pH值各为1.60和1.70,20℃水浴15 min是较适测定条件.检出限24.27 nmol·L-1.健康人血清亚硝酸盐含量为(10.91±2.38)μmol·L-1,95%分布范围为(6.24～15.57)μmol·L-1.结论:本法较为敏感、特异、简便,对一氧化氮的研究有一定价值.     

用荧光分光光度法测定组织和血液中一氧化氮

利用NO^－₂对4-羟基香豆素的荧光增强效应,建立了生物样本中NO荧光分光度测定法,其检测浓度范围为2×10^-5～2×10^-8 mol/L,采用该方法检测了大鼠大脑皮层和海马组织、细菌脂多糖(lipopolysaccharide,LPS)诱导的巨噬细胞培养上清和血清中NO含量.     

组织细胞一氧化氮含量测定的几种方法

一氧化氮(nitric oxide,NO)是一种新型的细胞信使分子,它在调节心血管系统、神经系统和免疫功能方面起着重要的作用.测定组织细胞 NO 的含量对于探讨NO 的生理功能具有重要的意义.该文简要介绍了应用化学发光法、微盘测定法、放射强度测定法和分光光度法检测组织细胞 NO 的含量.     

荧光标记电泳对透明质酸寡糖片段鉴定的方法建立

近年来,透明质酸寡糖片段（hyaluronan oligosaccharides, o-HA）的生物学活性引起国外学者的重视,因为o-HA具有一定的生物学活性,如参与免疫调节、刺激新生血管形成等.本研究建立一种经济、简便的ANTS（8-氨基奈-1,3,6-三磺酸）荧光标记电泳对透明质酸寡糖片段大小鉴定的新实验方法.实验原理为,ANTS能与糖分子发生还原反应,在反应时提供3个电子和1个荧光基团,通过高浓度PAGE分离,在特定波长下呈现颜色反应.采用酶消化法得到不同分子量大小的o-HA片段,测得不同片段大小的o-HA聚合度,分别与高效液相色谱（high-performance liquid chromatography, HPLC）和静电喷雾电离质谱（electrospray ionization mass spectrometry, ESI-MS）进行比较,结果吻合.研究提示,用荧光标记电泳法分析寡糖分子量,操作简单、设备低廉、灵敏度较高且检测速度快,是一种检测鉴定寡糖分子的较好方法.     

利用荧光探针直接测定线粒体活性氧的形成

目的与方法：根据荧光探针－还原型二氯荧光素（2‘,7‘-dichlorodihydrofluorescin,DCFH)可与活性氧（reactive oxygen species,ROS)反应生成荧光物一氧化型二氯荧光素（2‘,7‘-dichlorofluorecin,DCF)的原理,设计了利用荧光分光光度计直接定量检测线粒体活性氧生成并可观察在各种实验条件下线粒体活性氧产生动态变化的方法。结果与结论：线粒体在态4呼吸状态下,DCF的荧光强度随时间呈线性增加,表明活性氧以恒定速率产生。将荧光强度随时间变化的数据点拟合,线性回归直线斜率与活性氧产生的速率呈正比,测定中加入叠氮钠和丙二酸可分别使线粒体活性氧产生增加和减少。DCF荧光强度增加速率与线粒体浓度在一定范围内呈线性关系。复管实验表明重复性良好。     

应用HPLC进行流动荧光注射测定饲料中的色氨酸

应用ＨＰＬＣ进行流动荧光注射测定饲料中的色氨酸,比常规方法灵敏度高、快速。最佳测定条件为：激发波长３６５ｎｍ;发射波长４５０ｎｍ;流率是０．５ｍｌ／ｍｉｎ。     

大鼠脑组织中一氧化氮合酶测定

在含有一氧化氮合酶(NOS)底物左族精氨酸(L-Arg), 辅助因子还原性辅酶Ⅱ(NADPH)、四氢生物蝶呤(BH₄)、黄素单核苷酸(FMN), 黄素腺嘌呤二核苷酸(FAD)以及Ca²⁺、钙调蛋白等溶液中加入大鼠脑组织匀浆离心上清液, 组成酶反应体系. 37℃温育80min, 应用N-(1-萘基)-乙二胺、对氨基苯磺酸的重氮、偶氮反应测定酶反应体系中一定时间内NO代谢产物NO^-₂浓度变化, 建立一种简便的NOS活性测定方法. 反应体系最佳pH为7.4, K_m=0.1mmol/L, 体系内NO^-₂生成量与加入样品量之间有良好线性关系(r=0.998). 此方法简单、方便、重复性好, 批内CV为3.69%, 批间CV为5.16%. 10只健康大鼠脑组织中NOS活性为(39.61±7.64)nmol/(min·g).     

一种新型的DNA荧光染料—DAPI的光学特性及其应用

近年来,由于荧光显微镜的改进,荧光染料的不断增多以及免疫荧光技术的发展,荧光显微术已在细胞生物学的研究中广泛应用。荧光显微术用于研究细胞核和染色体已有较长的历史,不过目前除了哺乳类染色体分带荧光技术和孚尔根型荧光染色有所发展外,曾经使用过的一些核的荧光染色方法已应用不多。     

一种新型的DNA荧光染料-DAPI的光学特性及其应用

近年来,由于荧光显微镜的改进,荧光染料的不断增多以及免疫荧光技术的发展,荧光显微术已在细胞生物学的研究中广泛应用。荧光显微术用于研究细胞核和染色体已有较长的历史,不过目前除了哺乳类染色体分带荧光技术和孚尔根型荧光染色有所发展外,曾经使用过的一些核的荧光染色方法已应用不多。最近,一种新型的DNA荧光染     

Increase of nitrosative stress in patients with eosinophilic pneumonia

Background

Exhaled nitric oxide (NO) production is increased in asthma and reflects the degree of airway inflammation. The alveolar NO concentration (Calv) in interstitial pneumonia is reported to be increased. However, it remains unknown whether NO production is increased and nitrosative stress occurs in eosinophilic pneumonia (EP). We hypothesized that nitrosative stress markers including Calv, inducible type of NO synthase (iNOS), and 3-nitrotyrosine (3-NT), are upregulated in EP.

Methods

Exhaled NO including fractional exhaled NO (FE_NO) and Calv was measured in ten healthy subjects, 13 patients with idiopathic pulmonary fibrosis (IPF), and 13 patients with EP. iNOS expression and 3-NT formation were assessed by immunocytochemistory in BALf cells. The exhaled NO, lung function, and systemic inflammatory markers of the EP patients were investigated after corticosteroid treatment for 4 weeks.

Results

The Calv levels in the EP group (14.4 ± 2.0 ppb) were significantly higher than those in the healthy subjects (5.1 ± 0.6 ppb, p < 0.01) and the IPF groups (6.3 ± 0.6 ppb, p < 0.01) as well as the FE_NO and the corrected Calv levels (all p < 0.01). More iNOS and 3-NT positive cells were observed in the EP group compared to the healthy subject and IPF patient. The Calv levels had significant positive correlations with both iNOS (r = 0.858, p < 0.05) and 3-NT positive cells (r = 0.924, p < 0.01). Corticosteroid treatment significantly reduced both the FE_NO (p < 0.05) and the Calv levels (p < 0.01). The magnitude of reduction in the Calv levels had a significant positive correlation with the peripheral blood eosinophil counts (r = 0.802, p < 0.05).

Conclusions

These results suggested that excessive nitrosative stress occurred in EP and that Calv could be a marker of the disease activity.     

Effect of ammonium and nitrate application on the NO and N2O emission out of different soils

The effect of nitrate and ammonium application (0, 50, 100 and 150 mg N kg^-1 soil) was studied in an incubation experiment. Four Belgian soils, selected for different soil characteristics, were used. The application of both nitrate and ammonium caused an increase of the NO and N₂O emission. The NO production from nitrate and ammonium was found to be of the same order of magnitude. At low pH the NO production was found to be highest from nitrate, at higher pH values the production was found to be higher from ammonium. This seems to be the result of the negative effect of low pH on nitrification.The ANOVA analysis was carried out to separate the effect of the form of nitrogen, quantily of N applied and soil characteristics. The total production of NO was found to depend for 97% on the soil characteristics and for 3% on the quantity of N added. The total N₂O production depended for 100% on the soil characteristics.Stepwise regression analysis showed that the total NO production was best predicted by a combination of the factors CaCO₃ content and NH₄ ⁺ concentration in the soil. Total N₂O production was best described by a combination of CaCO₃, water soluble carbon (WSC) and sand-content.The N₂O/NO ratio was found to be highly variable, indicating that their productions react differently to changes in conditions, or are partly independent.It may be concluded that to NO and N₂O from soils both nitrification and denitrification may be equally important, their relative importance depending on local conditions such as substrate availability, water content of the soil etc. However, the NO production seems to be more nitrification dependent than the N₂O production. ei]{gnE}{fnMerckx}{edSection editor}     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.     

外源NO对NaHCO3胁迫下黑麦草幼苗光合生理响应的调节

采用营养液砂培方法,研究了外源一氧化氮(NO)对100 mmol/L NaHCO3胁迫下黑麦草幼苗叶片叶绿素含量、光合气体交换和叶绿素荧光参数、光能分配及叶黄素循环的影响。结果表明:(1)外施60μmol/L NO供体硝普钠(SNP)显著缓解了NaHCO3胁迫下叶绿素含量、净光合速率(Pn)、气孔导度(Gs)和气孔限制值(Ls)的下降及胞间CO2浓度(Ci)的升高,提高了光系统Ⅱ(PSⅡ)的潜在活性(Fv/Fo)、最大光化学效率(Fv/Fm)、实际光化学效率(ΦPSⅡ)和光化学猝灭(qP),降低了初始荧光(Fo)和非光化学猝灭(NPQ)。(2)NaHCO3胁迫下,外施SNP显著抑制了天线转换效率(Fv’/Fm’)的下降,降低了光系统间激发能分配的不平衡性(β/α-1)和天线热耗散的比例(D),提高了吸收光能中用于光化学反应的比例(P),而对PSⅡ反应中心的过剩光能(Ex)无明显影响。(3)外施SNP显著降低了NaHCO3胁迫下叶黄素循环库(V+A+Z)下降和叶黄素循环脱环氧化状态(A+Z)/(V+A+Z)上升的幅度。但SNP对NaHCO3胁迫的缓解效应可被NO清除剂血红蛋白(Hb)部分或完全地逆转,SNP的分解产物NaNO2处理对NaHCO3胁迫无明显改善。表明外源NO可能通过提高光化学效率,缓解了碱胁迫引起的光抑制对光合机构的破坏,从而提高黑麦草的光合效率。     

Mitochondria produce reactive nitrogen species via an arginine-independent pathway

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.     

Mitochondrial Trafficking in Neurons: A Key Variable in Neurodegeneration?

Mitochondria are the proximate target of a number of different neurotoxins. Typically, impairing of the key bioenergetic function of mitochondria by toxins is considered as the main mechanism of action. However, the effective maintenance of energy generation in neurons depends on the biogenesis, trafficking, and degradation of mitochondria in addition to the traditional bioenergetic functions. We have recently demonstrated that glutamate alters both the trafficking and morphology of mitochondria in primary neurons. In addition, several other potential neurotoxins, including nitric oxide and zinc, inhibit mitochondrial movement and, in some cases, alter morphology too. This suggests that some part of the action of neurotoxins might include the impairment of mitochondrial trafficking in neurons, with the resultant failure of local ATP delivery.