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1.
流感病毒神经氨酸酶抑制剂筛选模型的建立和应用 总被引:9,自引:0,他引:9
目的建立适用于高通量筛选的流感病毒神经氨酸酶(neuraminidase,NA)抑制剂筛选模型。方法从甲型及乙型流感病毒中制备神经氨酸酶,以2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid(MUNANA)作为底物,建立检测神经氨酸酶活性的荧光测定法及其抑制剂体外筛选方法,用高通量筛选系统对1 200个化合物与提取物进行初筛。结果神经氨酸酶酶促反应以pH 3.5,二价阳离子浓度为2~6 mmol·L-1及37℃孵育时酶活性最佳;甲、乙型流感病毒不同株神经氨酸酶的米氏常数(Km)的范围为(4.89~5.94) μmol·L-1;初筛发现12个化合物对流感病毒神经氨酸酶有可重复的抑制活性。结论优化了神经氨酸酶反应体系,建立的体外模型可用于抗甲、乙型流感病毒药物的高通量筛选及酶抑制动力学的研究。 相似文献
2.
目的建立以极化荧光为检测方法的高通量筛选技术,通过大规模筛选发现氧化型低密度脂蛋白受体-1(LOX-1)的天然配体。方法密度梯度超速离心获得正常人血中低密度脂蛋白(LDL),然后用CuSO4(5 μmol·L-1)修饰为氧化型低密度脂蛋白(oxLDL)。FITC标记hLOX-1,以受体(LOX-1)和配体(oxLDL)的相互作用为基础建立筛选模型,用极化荧光检测方法,在激发光485 nm、发射光525 nm,对3 200个样品进行高通量筛选,并用Z′因子值评价实验。结果Z′因子值为0.75,根据建立的基于极化荧光的高通量筛选实验方法,发现3个化合物与hLOX-1有较高的结合活性,IC50值小于31.6 μmol·L-1。结论极化荧光检测方法适合于高通量筛选技术,具有较高的稳定性、灵敏性和可重复性。 相似文献
3.
设计合成具有抗乙型肝炎病毒活性的非环核苷膦酸双L-氨基酸酯系列衍生物。以adefovir dipivoxil为先导化合物, 根据核苷L-氨基酸酯前药提高生物利用度及抗病毒活性的研究结果对先导化合物进行结构优化, 设计并合成一系列新型非环核苷膦酸双L-氨基酸类化合物, 确定其结构, 并通过测定其对HepG2 2.2.15细胞分泌的HBV-DNA的抑制作用评价其体外抗病毒活性。结果发现8个adefovir双L-氨基酸类化合物可显示不同程度活性, 其中化合物11的抗病毒活性最强、 选择性指数最高(EC50 0.095 2 μmol·L-1, SI 69523)。以上研究提示L-氨基酸酯策略可适用于非环核苷膦酸的前药修饰, 以期发现有效抗HBV药物。 相似文献
4.
目的设计、合成新的2-芳亚胺基-4-噻唑烷酮类化合物并研究其对一氧化氮合酶(NOS)的抑制活性。方法运用N-氯乙酰-1,2,3,4-四氢异喹啉或N-氯乙酰邻苯二甲酰亚胺与取代硫脲反应,简便地合成了15个新的2-芳亚胺基-4-噻唑烷酮类化合物,测试新合成的目标化合物的NOS抑制活性。结果和结论合成了15个新的2-芳亚胺基-4-噻唑烷酮类化合物,大部分反应收率大于65%。所有化合物的结构用1H NMR,IR,MS及元素分析进行了表征。初步药理筛选结果表明,部分化合物具有NOS抑制活性。 相似文献
5.
目的利用经药物诱导获得的mdr1基因高表达细胞株以及通过mdr1基因转染建立的稳定高表达细胞株,研究多药耐药肿瘤细胞对力达霉素(C-1027)的药物敏感性。方法构建mdr1重组真核表达质粒pcDNA3.1/mdr1,利用脂质体转染技术,获得mdr1高表达HepG2肝癌细胞。经RT-PCR、细胞荧光免疫化学及罗丹明外排实验,鉴定了细胞的mdr1表达水平和药物外排活性。MTT方法测定敏感细胞及相对应的多药耐药细胞对力达霉素等多种抗肿瘤药物的药物敏感性。结果mdr1稳定转染细胞株HepG2/mdr1、多药耐药KBv200细胞和MCF-7/ADR细胞对力达霉素的IC50值分别为(0.020±0.011) nmol·L-1,(0.24±0.20) nmol·L-1和(0.028±0.011) nmol·L-1。相对于各自的敏感细胞,多药耐药细胞HepG2/mdr1,KBv200和MCF-7/ADR对力达霉素的抗药倍数分别是1.3,6.8和1.6倍,对阿霉素的抗药倍数分别是8.8, 37.2和181.3倍,对紫杉醇的抗药倍数分别是40.3, 336.8和49.2倍。结论 mdr1高表达的多药耐药肿瘤细胞对力达霉素仍高度敏感,未表现出抗药性。 相似文献
6.
年龄及人参皂甙 Rg1 对大鼠大脑皮层 NO 释放的影响 总被引:14,自引:0,他引:14
探讨了NO与衰老的关系及与Rg1抗衰老机制的关系。用Griess法,3H-L-精氨酸转化法分别研究大鼠大脑皮层细胞NO含量及NOS活性,观察其随龄变化及人参皂甙Rg1(Rg1)对老年鼠NO及NOS的影响。实验表明,成年鼠(9月龄)与青年鼠(3月龄)NO含量及NOS活性无显著性差异。老年鼠(27月龄)脑皮层NO含量明显高于青年鼠和成年鼠,NOS活性也明显增高。老年鼠给予Rg1后可显著减少大脑皮层NO含量和降低NOS活性。结果提示,NO与衰老关系密切,Rg1的抗衰老作用与其对NOS活性的抑制有关。 相似文献
7.
目的研究非甾体抗炎药艾拉莫德(T-614)对脂多糖(LPS)刺激的大鼠肺泡巨噬细胞系(NR8383)前炎症反应因子TNFα基因表达、蛋白合成的影响及其对核因子κB(NF-κB)的作用。方法体外培养NR8383经T-614(13.4,26.7及53.4 μmol·L-1)处理,LPS刺激后应用酶联免疫吸附法(ELISA)检测细胞上清液中TNFα的水平,半定量逆转录聚合酶链式反应(RT-PCR)检测TNFα mRNA水平,ELISA法检测NF-κB的活性。结果T-614对LPS诱导的NR8383细胞TNFα mRNA水平和蛋白水平的上调有显著抑制作用,对NF-κB的转录活性也有抑制作用。结论 T-614可能通过抑制LPS诱导的NR8383的NF-κB活性而降低TNFα的产生。 相似文献
8.
南重楼Paris vietnamensis活性物质的分离与鉴定 总被引:1,自引:0,他引:1
目的研究南重楼Paris vietnamensis (Takht.)中具有细胞毒活性的甾体皂苷类化学成分。方法采用柱色谱进行分离纯化,通过光谱分析和理化方法鉴定化合物结构,并进行体外细胞毒活性筛选。结果从南重楼中分离得到11个甾体皂苷类化合物,化合物1结构为:3β,5α,6α-三羟基-7(8)-烯-异螺甾烷醇-3-氧-β-D-葡吡喃糖基(1→3)[α-L-鼠李吡喃糖基(1→2)]-β-D-葡吡喃糖苷(1),命名为南重楼皂苷A。化合物2~11分别为:25(R)-薯蓣皂苷元-3-氧-α-L-阿拉伯呋喃糖基(1→4)-β-D-葡吡喃糖苷(2);25(R)薯蓣皂苷元-3-氧-α-L-鼠李糖吡喃基(1→2)-β-D-葡吡喃糖苷(3);25(R)薯蓣皂苷元-3-氧-α-L-阿拉伯呋喃糖基(1→4)[α-L-鼠李吡喃糖基(1→2)]-β-D-葡吡喃糖苷(4);25(R)薯蓣皂苷元-3-氧-β-D-葡吡喃糖基(1→3)[α-L-鼠李吡喃糖基-(1→2)]-β-D-葡吡喃糖苷(5);25(R)薯蓣皂苷元-3-氧-α-L-鼠李吡喃糖基(1→4)-α-L-鼠李吡喃糖基(1→4)[α-L-鼠李吡喃糖基(1→2)]-β-D-葡吡喃糖苷(6);25(R)偏诺苷元-3-氧-α-L-阿拉伯呋喃糖基(1→4)-β-D-葡吡喃糖苷(7);25(R)偏诺苷元-3-氧-α-L-鼠李吡喃糖基(1→2)-β-D-葡吡喃糖苷(8);25(R)偏诺苷元-3-氧-α-L-阿拉伯呋喃糖基(1→4)[α-L-鼠李吡喃糖基(1→2)]-β-D-葡吡喃糖苷(9);25(R)偏诺皂苷元-3-氧-β-D-葡吡喃糖基(1→3)[α-L-鼠李吡喃糖基(1→2)]-β-D-葡吡喃糖苷(10);25(R)偏诺苷元-3-氧-α-L-鼠李吡喃糖基(1→4)-α-L-鼠李吡喃糖基(1→4)[α-L-鼠李吡喃糖基(1→2)]-β-D-葡吡喃糖苷(11),体外筛选表明部分甾体皂苷类化合物具有细胞毒作用。结论化合物1为一新化合物,化合物2,3,6~11均为首次从南重楼中获得。化合物3,4,6,8具有对肝癌HepG2和胃癌SGC-7901的细胞毒活性。 相似文献
9.
目的 建立一个高通量筛选防治动脉粥样硬化先导化合物的体外细胞模型。方法 克隆HIF-1α低氧反应元件(hypoxia response element,HRE)至荧光素酶报告基因表达载体pGL3-Enhancer,构建荧光素酶表达载体pGL3-HIF-1α-HRE,转染人单核细胞THP-1并筛选稳定表达细胞株THP-1-HIF-1α-HRE。结果 Real Time-PCR检测表明低氧培养可以有效上调THP-1-HIF-1α-HRE细胞HIF-1α蛋白表达和荧光素酶活性,而洛伐他汀和姜黄素预处理可以有效抑制低氧引起的THP-1-HIF-1α-HRE细胞内HIF-1α蛋白表达及荧光素酶活性。结论 成功建立了筛选抗动脉粥样硬化的先导化合物体外高通量细胞模型THP1-HIF-1α-HRE。 相似文献
10.
新的核苷类化合物β-L-D4A的化学合成及体外抗HBV作用 总被引:4,自引:0,他引:4
目的以D型谷氨酸为原料,通过一系列化学转化,合成了新的核苷类化合物β-L-D4A,并初步探索其体外抗HBV作用。方法合成β-L-D4A,用红外光谱、核磁共振氢谱和质谱确证目标化合物的结构,以2.2.15细胞(HepG2细胞进行HBV基因组转染后所得)培养为基础,Southern印迹法检测不同浓度化合物体外抑制HNV DNA复制作用,并求出50%抑制的药物浓度,即EC50。以四噻唑蓝(MTT)比色分析法检测不同浓度药物的细胞毒性,求出IC50。结果化合物β-L-D4A经红外光谱、核磁共振氢谱和质谱确证;2.2.15细胞培养上清液病毒DNA的Southern印迹、自显影结果显示病毒的抑制呈明显的浓度依赖性,计算出EC50为0.2 μmol·L-1,胞内DNA的Southern印迹、自显影显示类似的结果;细胞毒性实验显示IC50为200 μmol·L-1。结论体外实验显示β-L-D4A具有明显的抑制病毒DNA复制作用,且无明显的细胞毒性,TI值为1 000,高于临床用Lamivudine (750),有望开发为临床抗HBV用药。 相似文献
11.
高糖抑制牛主动脉内皮细胞诱导型和结构型一氧化氮合酶的表达 总被引:3,自引:1,他引:2
目的:研究高糖对新生小牛主动脉内皮细胞(BAEC)一氧化氮合酶(NOS)表达的影响。方法:BAEC培养并传代于含正常葡萄糖(5.5mmol·L~(-1),NGBAEC),高糖(25 mmol·L~(-1),HG-BAEC)或高渗(葡萄糖5.5 甘露醇19.5 mmol·L~(-1),Mann-BAEC)的无酚红M1640培养基.Griess反应检测脂多糖(LPS)诱导的一氧化氮(NO)产生.Westem blot法检测结构型NOS(ecNOS)及诱导型NOS(iNOS)表达。结果:LPS(0.25-2 mg·L~(-1))剂量依赖性刺激BAEC产生NO,并在LPS 1 mg·L~(-1)达峰值。高糖显著抑制LPS诱导的NO产生(亚硝酸μmol·L~(-1):HG-BAEC 43±8,vs NG-BAEC 71±11,Mann-BAEC 70±9,n=4,P<0.01)。同样,与NG-和Mann-BAEC相比,HG-BAEC iNOS表达下降39.9%和39.3%,ecNOS表达下降28%和24%,而NG-与Mann-BAEC之间,LPS诱导的NO产量和iNOS和ecNOS的表达无差别。结论:高糖抑制BAEC NO的释放,与NOS的低表达有关。 相似文献
12.
Altered L-arginine/nitric oxide synthase/nitric oxide pathway in the vascular adventitia of rats with sepsis 总被引:1,自引:0,他引:1
Jia YX Pan CS Yang JH Liu XH Yuan WJ Zhao J Tang CS Qi YF 《Clinical and experimental pharmacology & physiology》2006,33(12):1202-1208
1. In recent studies, the vascular adventitia has been established as an important source of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production, even more powerful than the media in response to certain inflammatory factors, such as lipopolysaccharide (LPS). The adventitia has an independent L-arginine (L-Arg)/NOS/NO pathway and is involved in the regulation of vascular function. In the present study, we explored the changes in and the pathophysiological significance of the L-Arg/NOS/NO pathway in the adventitia of rats with sepsis. 2. Sepsis was induced by caecal ligation and puncture in order to observe changes in L-Arg transport, NOS gene expression and activity and NO generation in the vascular adventitia to determine the mechanism of activation of the L-Arg/NOS/NO pathway. 3. Severe sepsis resulted in severe disturbance of haemodynamic features, with decreased mean arterial blood pressure, brachycardia and inhibited cardiac function (decreased left ventricular +/-dP/dt(max)). Left ventricular end-diastolic pressure was elevated threefold (P < 0.01) under anaesthesia. Rats with sepsis showed severe glucopenia and lacticaemia. Plasma levels of the inflammatory factors macrophage chemoattractant protein-1 and interleukin-8 were increased five- and 29-fold, respectively (P < 0.01). 4. In the adventitia of the thoracic and abdominal aortas, the L-Arg/NO pathway was similarly characterized: the uptake of [(3)H]-L-Arg was Na(+) independent, with the peak occurring at approximately 40 min incubation. Total NOS activity was largely calcium independent (> 90%). The V(max) of L-Arg transport in the sepsis group was increased by 83.5% (P < 0.01), but the K(m) value was not significantly different compared with controls. 5. The mRNA levels of cationic amino acid transporter (CAT)-1 and CAT-2B in the sepsis group were increased by 86 and 62%, respectively (both P < 0.01). Inducible NOS activity was increased 2.8-fold compared with controls (P < 0.01) and iNOS mRNA levels were elevated approximately sixfold (P < 0.01). The NO levels in the plasma and incubation media (incubation for 40 min) in the sepsis group were increased by 144 and 273%, respectively (both P < 0.01). 6. The Arg/NOS/NO pathway was activated in the vascular adventitia of rats with sepsis shock. The L-Arg/NOS/NO pathway in the aortic adventitia may play an important role in the pathogenesis of sepsis and septic shock. 相似文献
13.
目的研究果糖二磷酸钠镁对缺血突触体游离钙浓度及一氧化氮合酶活性的影响,以探讨其保护脑缺血的作用机制。方法以相分离法制备正常大鼠脑突触体,氧糖剥夺培养建立缺血突触体模型,加入1.3 mmol·L-1 SMFD,4.0 mmol·L-1 FDP与之培养60 min后,测定突触体内游离钙浓度和一氧化氮合酶活性。结果1.3 mmol·L-1 SMFD可明显降低缺血突触体内游离钙浓度,降低一氧化氮合酶活性,其作用强于4.0 mmol·L-1 FDP。结论SMFD保护脑缺血的作用机制可能与其阻止脑缺血后细胞内钙超载,抑制一氧化氮合酶活性有关。 相似文献
14.
目的:研究dBcAMP诱导分化成熟后的星状胶质细胞内脂多糖诱导型一氧化氮合酶活力的诱导表达情况。方法:采用荧光分光光度法和胍胺酸生成实验考察星状胶质细胞内一氧化氮合酶的活力,并通过免疫细胞化学和光学显微镜考察细胞的分化情况。结果:经dBcAMP诱导后,星状胶质细胞分化成星型细胞,GFAP表达增强,dBcAMP显著增强LPS诱导产生的亚硝酸盐水平的提高;dBcAMP/LPS共同诱导24h后,使NOS 相似文献
15.
Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications. 总被引:2,自引:0,他引:2
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A. García-Pascual G. Costa A. Labadia K. Persson D. Triguero 《British journal of pharmacology》1996,118(4):905-914
1. To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO-mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2. NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg-1 protein min-1): urethra (33 +/- 3.3), detrusor (13.1 +/- 1.1) and ureter (1.5 +/- 0.2). No activity was detected in the particulate fraction of any region. 3. NOS activity was dependent on Ca(2+)-calmodulin and required exogenously added NADPH and tetrahydrobyoptein (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4. NOS activity was inhibited to a greater extent by NG-nitro-L-arginine (L-NOARG) and its methyl ester (L-NAME) than by NG-monomethyl-L-arginine (L-NMMA), while 7-nitroindazole (7-NI) was a weak inhibitor and L-cannavine had no effect. 5. Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin-sensitive manner, suggesting feedback inhibition of NOS by NO. 6. EFS induced prominent NO-mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L-NOARG = L-NAME > 7-NI > L-NMMA. 7. In conclusion, we have demonstrated the presence of NO-synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO-mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely. 相似文献
16.
目的观察 NO合酶( NO synthase,NOS)抑制剂左旋硝基精氨酸(NG-nitro-L-arginine,NOArg)和 NO合成前体左旋精氨酸(L-arginine,L-Arg)对去甲吗啡(normorphine,NM)在离体豚鼠回肠上依赖性耐受性产生的影响,研究NO在阿片类药物依赖性耐受性产生中的作用。方法以离休豚鼠回肠为实验对象,4℃孵育6 h, 24 h后,分别测定纳洛酮(naloxone,Nx)与氯化乙酰胆碱(acetylcholine,Ach)引起的收缩高度比值,作为依赖性产生的指标。在耐受性产生实验中,分别测定回肠空白组和药物组4℃孵育3h后对NM抑制电刺激收缩的剂量反应曲线。结果回肠分别经NM,NM加NOArg,NM加L-Arg中孵育6h后,纳洛酮均可使其产生戒断收缩,且两组与Ach的收缩高度比值差异无显著性(n=6), P>0.05。而孵育24 h后,NM,NM加 L-Arg组收缩高度比值分别为 0. 89±0. 02, 0. 90±0. 02, P> 0. 05,而NM加NOArg药物组对纳洛酮的戒断收缩反应明显减弱,收缩高度比值为 0.47± 0.05(n= 6, P< 0.01)。可见NM加NOA? 相似文献
17.
18.
We explored the possible role of the nitric oxide (NO) system in hydroxyl radical (*OH) generation induced by carbon monoxide (CO) poisoning in rat striatum by means of microdialysis with the use of NO synthase (NOS) inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA), as well as L-arginine (L-Arg; the NOS substrate) and D-arginine (D-Arg). The CO-induced *OH generation was suppressed by both L-Arg and D-Arg. It was also suppressed by L-NAME, which inhibits generation of reactive oxygen species (ROS) via neuronal NOS (nNOS) and inducible NOS, but not via endothelial NOS. In contrast, L-NMMA, which inhibits only ROS generation via inducible NOS, potentiated the *OH generation. L-Arg completely reversed the L-NAME effect and partly reversed the L-NMMA effect. D-Arg reversed the L-NAME effect more potently than did L-Arg, resulting in much more *OH generation than was observed with CO alone, and also potentiated the L-NMMA effect. On the other hand, W-7, an antagonist of calmodulin, which is critical for nNOS activity, had no effect on the CO-induced *OH generation. These findings suggest that complex mechanisms operate in *OH generation in rat striatum upon CO poisoning and that the NO system might not be included among those mechanisms. 相似文献
19.
目的:研究牛心内膜内皮细胞(BEEC)腺苷三磷以磷酸酶(apyrase)的特性并比较牛心内膜及血 皮细胞的胞外腺苷酸酶活性。方法:以反相HPLC测定核苷酸含量,以磷离子释放法检测apyrase活性。结果:叠氮钠10mmol.L^-1和氟化钠20mmol.L^-1分别抑制BEEC apyrase51%及38%活性,而Na^+/K^+-ATP酶抑制剂哇巴因则对BEED apyrase活性无影响。过氧化 相似文献
20.
目的:建立人类类固醇5αlphaⅡ型还原酶(hSRD5A2)体外药物筛选新模型,并用于中药提取物的筛选。方法:在体外建立hSRD5A2抑制剂模型,hSRD5A2、睾酮(T)和还原型辅酶Ⅱ(NADPH)一起组成了一个微量反应系统,对底物浓度、NADPH、hSRD5A2、温度和时间进行优化,并利用建立的模型筛选中药提取物。结果:当固定底物、NADPH、hSRD5A2的浓度分别为0.02μmol·L-1、0.8mmol·L-1、0.05u·mL-1时,在37℃反应60min,hSRD5A2达到了最大活性;芒果苷有显著抑制hSRD5A2活性的作用。结论:本研究建立的模型是一种简单可靠的筛选hSRD5A2抑制剂的方法;芒果苷可能成为一种治疗良性前列腺增生的药物。 相似文献