软体动物的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,参与调节软体动物的嗅觉、运动、取食、机体防御及学习行为。本文从生理、生化、形态定位以及信号转导几方面综述了有关软体动物一氧化氮及其合酶的最新研究进展。     

一氧化氮合酶的研究进展

一氧化氮是由Ｌ－精氨酸和氧分子在一氧化氮合酶及其辅因子ＮＡＤＰＨ、ＦＡＤ、ＦＭＮ、ＣａＭ和ＢＨ４催化作用生成的;ＮＯＳ分为原生型和诱生型ＮＯＳ,原生型ＮＯＳ活性依赖于胞浆内Ｃａ＾２＋水平,诱生型ＮＯＳ是Ｃａ＾２＋／ＣａＭ非依赖性酶,其活性开关是胞内ｎＮＯＳ ｍＲＮＡ水平,ＮＯＳ可在多个水平被调节;ＮＯＳ可能在心血管疾病的发病中起重要作用。     

一氧化氮合酶的作用机制

一氧化氮合酶的作用机制赵慧卿（安徽医科大学化学教研室,合肥２３００３２）关键词一氧化氮一氧化氮合酶８０年代以来,人们发现一氧化氮在许多生理过程中起着十分重要的作用［１］。在血管内皮细胞中,一氧化氮可激活可溶性鸟苷酸环化酶（ｓＧＣ）,通过升高环鸟苷酸水...     

一氧化氮合酶的若干研究进展

一氧化氮合酶(NOS)是一氧化氮(NO)生物学与医学研究的重要内容.近年来,对NOS酶本质及其生化与分子生物学特性甚至某些分子遗传学方面的认识都在迅速发展和深化.研究表明,干预NOS-NO途径的某些环节,如酶激活、NO合成、释放与转运甚至有关酶的编码基因及其表达,将为某些临床问题的解决提供新的思路和手段．     

一氧化氮合酶基因缺陷动物模型的应用研究进展

一氧化氮合酶基因缺陷动物模型的应用研究进展马子敏何子安（军事医学科学院卫生学环境医学研究所,３０００５０天津）关键词一氧化氮一氮化氮合酶一氧化氮合酶（ＮＯＳ）广泛存在于机体。它主要有三个类型：神经型ＮＯＳ（ｎＮＯＳ）、内皮型ＮＯＳ（ｅＮＯＳ）、诱导型...     

诱生型一氧化氮合酶基因启动子的结构及其调控

鼠类ＮＯＳ２基因５′端１．７ｋｂ序列几乎包含了所有的顺式作用元件,其中ＮＦκＢ结合位点,ＩＲＦ－ＲＥ,ＣＡＡＴｂｏｘ和ＧＡＳ４等元件对该在的诱导性转录调控至关重要,人ＮＯＳ２基因５′端３．７ｋｂ范围与鼠类有相似的调控元件,但该区域仅有基础启动子活性,其诱导性调控元件在－３．７ｋｂ的上游,其中ＮＦκＢ结合位点着关键的作用。     

金黄色葡萄球菌对小鼠产生一氧化氮及一氧化氮合酶的影响

目的 :探讨金黄色葡萄球菌对小鼠产生一氧化氮 (NO)及一氧化氮合酶 (NOS)的影响 ,以进一步研究 NO及 NOS在抗感染免疫中的作用。方法 :将不同剂量的金黄色葡萄球菌注入小鼠腹腔 ,10 d后取小鼠血清和腹腔巨噬细胞培养上清 ,用硝酸还原酶法检测其 NO的含量 ,同时测定血清中 NOS的水平及抗金黄色葡萄球菌抗体的效价。结果 :金黄色葡萄球菌注射小鼠后 ,血清中 NO及 NOS的水平明显高于对照组 (P<0 .0 1) ,各组间两两比较亦差异有显著性 (P<0 .0 1)。腹腔巨噬细胞培养上清 NO的水平明显高于对照组 (P<0 .0 1) ,但不同剂量实验组之间差异无显著性 (P>0 .0 5)。结论 :金黄色葡萄球菌可引起小鼠血清中 NO、NOS升高 ,NO及 NOS可能在抗微生物感染免疫中起着重要的作用     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

诱导型一氧化氮合酶与疾病

炎症是众多疾病如自体免疫紊乱、神经退行性病变、心血管疾病和癌症发展的病理机制,诱导型一氧化氮合酶在炎症过程中被诱导表达,产生过量的一氧化氮,引发炎症级联反应,进而导致以上多种疾病发生。抑制诱导型一氧化氮合酶表达在体内体外实验及临床使用中均体现抗炎效果和症状改善。本文综述了诱导型一氧化氮合酶在炎症过程中诱导表达及与各类重大疾病联系的最新进展,并展望了诱导型一氧化氮合酶抑制剂作为抗炎治疗策略的前景。     

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).     

甲醛炎性痛增强大鼠海马NOS表达和NO生成

目的：观察甲醛炎性痛过程中大鼠痛行为、海马一氧化氮合酶（NOS）活性及一氧化氮（NO）含量的变化以及变化的时程及区域特征。方法：采用辐射热甩尾法测定大鼠痛阈变化;采用NADPH—d组织化学法和硝酸还原酶法分别测定大鼠海马NOS表达和No含量。结果：皮下注射甲醛溶液后,大鼠出现伤害性感受反应及痛阈降低。注射甲醛后6h,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加。海马NO含量亦显著增加;注射甲醛后12h时这些改变最为显著,48h时恢复至对照组水平。结论：甲醛炎性痛可诱导海马NOS活性增强及NO生成增多．这种改变可发生在海马各区．并具有一定的时程特征。     

诱导型一氧化氮合酶介导β淀粉样蛋白在体神经毒性

目的：探讨一氧化氮合酶（NOS）及一氧化氮（NO）在β淀粉样蛋白（Aβ）神经毒性和Alzheimer病（AD）发病机制中的介导作用。方法：应用行为学及病理学方法,观察海马注射Aβ1－40对大鼠Y迷宫学习记忆的影响及对局部神经元的损伤作用;观察特异性诱导型一氧化氮合酶（iNOS)抑制剂胍氢酶（AG）及特异性神经元型一氧化氮合酶(nNOS)抑制剂7－硝基吲哚（7－NI）腹腔注射对海马内注射Aβ1－40神经毒性的干预,结果：海马注射Aβ1－40后,大鼠Y迷宫学习记忆能力及海马局部神经元明显受损,特异性iNOS抑制剂AG能够阻止Aβ1－40海马注射对大鼠学习记忆和局部神经元的损伤作用,而特异性nNOS抑制剂7－NI无此干预效应。结论：iNOS/NO参与了在体条件下对Aβ神经毒性的介导,在AD发病机制中具有重要作用。     

诱导型一氧化氮合酶的激活与血压的关系

本实验旨在探讨诱导型一氧化氮合酶（iNOS)的激活与血压之间的关系,三组SD大鼠分别静脉输注不同浓度（0．3％,4％及8％）NaCl溶液以使其处于不同的血压水平,运用同位素标记的L－精氨酸转换成L－Citrulline 的转换率变化及Greiss反应,分别测定不同血压时iNOS的活性及NO的生成量,另四组大鼠包括正常Wistar,正常SD,高盐诱导的高血压（NaHR)及自发性高血压大鼠（SHR）,经测定血压后,取主动脉血管并以Western印迹印交法测定其iNOS蛋白水平,结果表明,血压较低时,SD大鼠iNOS活基本没有改变,而在输入4％和8％NaCl并处于较高血压水平的SD大鼠,其iNOS活性及NO生存均明显升高,。此外Western 印迹表明,两种高血压大鼠主动脉组织iNOS蛋白水平均较正常Wistar及正常SD大鼠高,密度扫描表明,NaHR及SHR主动脉组织iNOS蛋白分别较正常SD大鼠及正常Wistar大鼠升高149％及261％,这一结果提示,诱导型一氧化氮合酶是血液动力学调控的重要组成部分,尤其是在血压处于较高水平时,iNOS具有重要的代偿调节作用,除细胞因子,细菌产物等之外,血压也是调节iNOS表达及活性的重要因素之一。     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

诱导型一氧化氮合酶对内毒素休克小肠微循环的影响

采用静脉注射脂多糖(1ipopolysaccharide,LPS)的方法建立小鼠内毒素休克模型,探讨内毒素休克时小肠微循环的变化以及诱导型一氧化氮合酶(iNOS)对小肠微循环的影响。实验过程中连续监测小鼠平均动脉血压(mean afterial pressure,MAP)变化情况。利用FTTC标记红细胞和活体显微镜方法直接观察并计算小鼠小肠绒毛尖端小动脉和毛细血管内红细胞的流速和流量,并观察敲除小鼠iNOS基因和选择性iNOS抑制剂S-methylthiourea sulfate(SMT)对实验过程中小肠微循环的影响。结果显示,对于野生型小鼠,应用SMT处理和敲除iNOS基因对基线的MAP、小肠绒毛尖端小动脉和毛细血管的红细胞流速和流量没有显著性差别。给予LPS后,小鼠的MAP进行性下降。给予LPS前,应用SMT和敲除小鼠iNOS基因可以显著提高MAP：给予LPS后,小鼠小肠绒毛尖端小动脉和毛细血管内红细胞流速和流量显著下降。给予LPS前,应用SMT和敲除小鼠iNOS基因可以显著提高小肠绒毛尖端小动脉和毛细血管的红细胞流速和流量。结果表明,iNOS在内毒素休克小肠微循环衰竭的过程中发挥重要作用。一能性     

Production of nitric oxide under Ultraviolet-B irradiation is mediated by hydrogen peroxide through activation of nitric oxide synthase

Excised leaves of kidney bean (Phaseolus vulgaris) were used to investigate the mechanism of NO generation under UV-B stress. We showed that two signaling molecules, NO and H₂O₂, were produced in the irradiated leaves. NO release was blocked by LNNA, an inhibitor of NOS. Application of CAT (EC 1.11.1.6) not only effectively eliminated H₂O₂ in the leaves, but also inhibited the activity of NOS and the emission of NO. In contrast, treatment with exogenous H₂O₂ increased both of those events. Therefore, we suggest that, under UV-B stress, NO production is mediated by H₂O₂ through greater NOS activity.     

Alternative pathways of nitric oxide formation in Lactobacilli: Evidence for nitric oxide synthase activity by EPR

The study of the ability of Lactobacillus plantarum 8P-A3 to synthesize nitric oxide (NO) showed that this strain lacks nitrite reductase. However, analysis by the EPR method revealed the presence of nitric oxide synthase activity in this strain. Like mammalian nitric oxide synthase, lactobacillar NO synthase is involved in the formation of nitric oxide from L-arginine. L. plantarum 8P-A3 does not produce NO in the denitrification process. The regulatory role of NO in symbiotic bacteria is emphasixed.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K_m for arginine 2.8, 2.0 and 2.3 μM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Neural tube closure depends on nitric oxide synthase activity

Neural tube (NT) closure is a multifactorial process that involves yet unresolved molecular mechanisms. It had been shown previously that high levels of nitric oxide (NO) block the process of NT closure in the chick embryo by inhibiting methionine synthase (MS). The MS inhibition and its effect on NT closure could be alleviated by folic acid, suggesting the involvement of the folate-methionine pathway in the process. Here we test the hypothesis that endogenous nitric oxide synthase (NOS) activity regulates the MS activity required in the process of NT closure. The experiments described here reveal that NOS activity per se, is indeed critical for NT closure in the chick embryo. Inhibition of NOS activity with either 2,4-diamino-6-hydroxypyrimidine (DAHP), which blocks biosynthesis of the NOS co-factor tetrahydrobiopterin (BH4), or with calmidazolium, which blocks calcium-calmodulin binding to NOS, resulted in reduced MS activity and consequently ablated NT closure. Addition of BH4 or the calcium ionophore A23187 restored NOS and MS activities, resulting in NT closure. The results described here imply that NOS and MS activities can serve as functional markers in this developmental process as they are essential in the process of NT closure.