Biochemical Analysis Core

生化分析核心

• 批准号: 7700643
• 负责人: Juan C Romero
• 金额: $ 17.44万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7700643
• 关键词: 3-nitrotyrosine; ANG gene; Acetylcholinesterase; Actins; Alkanesulfonates; Analysis of Variance; Angiotensin I; Anions; Antibodies; Antioxidants; Back; Binding; Biochemical; Biochemical Reaction; Biological; Biological Assay; Blood Pressure; Buffers; Calcium Channel Blockers; Captopril; Cell Nucleus; Cells; Chemicals; Chronic; Color; Complex; Condition; Copper; Count; Creatinine; Cytolysis; DNA; Densitometry; Detection; Dithionitrobenzoic Acid; Endothelin; Enzyme Immunoassay; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Ethanol; Exhibits; Figs - dietary; Film; Fluorescence; Fluorescence Microscopy; Fluorescent Dyes; Freeze Drying; Freezing; Furuncles; Gamma counter; Gel; Generations; Genus Capra; Glutathione Disulfide; Glutathione Reductase; Goat; Guanidinium Chloride; Hexanes; Homidium Bromide; Hour; Hydrolysis; Hypertension; Ice; Immune Sera; In Situ; Incubated; Instruction; International; Isoprostanes; Kidney; Label; Left; Link; Lipoprotein Binding; Liquid substance; Literature; MCO chemical; Measurement; Measures; Membrane; Methanol; Methods; Milk; Monoclonal Antibodies; Mus; NADP; NADPH Oxidase; Neuro-Oncological Ventral Antigen 2; Nitrogen; Organic solvent product; Oryctolagus cuniculus; Oxidative Stress; Oxides; Oxis; Plasma; Potassium; Potassium Hydroxide; Principal Investigator; Production; Prostaglandins; Protease Inhibitor; Protein Analysis; Proteins; Pyridium; Pyroxylin; RENBP gene; Rate; Rattus; Reaction; Reactive Oxygen Species; Reader; Reading; Reagent; Relative (related person); Renal function; Renin; Renin-Angiotensin System; Research; Sampling; Signal Transduction; Sodium; Solutions; Staining method; Stains; Standards of Weights and Measures; Students; Sulfhydryl Compounds; Superoxides; Temperature; Testing; Time; Tissue Sample; Tissues; Tracer; Tube; Tyrosine; Uric Acid; Urine; Variant; Vendor; Water; Western Blotting; angiogenin; base; bathocuproine; cyclooxygenase 1; cyclooxygenase 2; day; dihydroethidium; ethyl acetate; flame photometry; fluoroform; gel electrophoresis; hemodynamics; human AKAP13 protein; in vivo; inhibitor/antagonist; metaphosphoric acid; nitration; oxidation; polyvinylidene fluoride; programs; reconstitution; response; size

by Dia-Sorin. The assay begins with samples (zero and incubated) and standards being incubated for 3 hours at room temperature in antibody coated tubes with I125 tracer added. After removing the elluent, antibody coated tubes are counted in a gamma counter and sample values obtained by comparing those of samples to those of the standard curve. The zero sample is then subtracted from the incubated sample to determine the amount of angiotensin I generated (Figsl ,2)(3,4). In mice, due to PHS 398/2590 (Rev.09/04, Reissued 4/2006) Page 315 Continuation Format Page Principal Investigator/Program Director (Romero,J.Carlos): Romero, J. Carlos COTG C problems with measuring PRA, we will instead measure PRC. Briefly for PRC generation we will use the methods of Carretero and Beierwaltes (5,6). This involves incubating our mouse samples for 2 hours at 37 ¿C with nephrectomized rat substrate in the presence of a peptidase inhibitor. To terminate the reaction, samples are boiled for 10 minutes then centrifuged for 15 minutes at 2000 rpm's. The supernatant is then removed and stored at -20 ¿C until assayed using the same kit as used for PRA measurement (Dia-Sorin). Anaiotensin II plasma and tissue: To extract Ang II from the tissue, we will first smash the tissue at-80'|0 C, then homogenize in cold MeOH. Samples are then centrifuged at 20,000 G's, the MeOH supernatant sample removed, lyophilized, and stored at -80¿C until reconstituted into assay buffer for extraction. Reconstituted tissue samples and plasma sampleswill use the same methods for extraction and assaying. Basically samples will be extracted on a pre-washed phenyl extraction column washed with dH2O and eluted with MeOH. The MeOH is removed by lyophilization and the samples reconstituted in assay buffer. The Enzyme Immunoassay (EIA) kit used is provided by SPI-Bio and distributed by Cayman Chemical. It involves an incubation of sample and standard with a monoclonal antibody immobilized to the wells of a 96 well plate. After immunological reaction the wells are washed and the trapped ANG II molecule is covalently linked back to the antibody complex with gluteraldehyde. After washing and denaturing the bound ANG II can react with the acetylcholinerase-labelled antibody (tracer). After washing to remove unbound tracer, the tracer bound to the ANG II complex reacts with Ellman's reagent to slowly develop a yellow color that is read on a spectrophotometer plate reader at the wavelength of 405 nm. Readings of samples are compared to those of standards to obtain a value. This value is given as pg per mg protein for the tissue samples and pg/ml for the plasma samples. This assay exhibits a 2-7% intra- and inter-assay coefficient when measuring from 20-100 pg of Ang ll/ml and 2-5% variation when estimating 2-5 pg of Ang II (Fig 2) (4,7,8,9). Nitrotvrosine - tissue: The principle of this assay is based on the fact that NO is the only biological molecule produced in high enough concentration to out-compete SODfor superoxides to form ~OONO. This compound modifies tyrosine in proteins to create nitrotyrosines leaving a marker detectable in vivo. Monoclonal antibodies to nitrotyrosine can then be used to determine immunohistochemically the~OONO- dependent tyrosine nitration. Kidney tissue froze in liquid nitrogen will be homogenized in 20 mM Hepes, pH 7.5, containing protease inhibitors, and will be centrifuged 600 xg to remove cellular debris. Gel electrophoresis analyses for nitrotyrosine residues will be performed on the supernatant as previously described using the anti-nitrotyrosine monoclonal antibody (Cayman,Ann Arbor) as the primary antibody. Following size separation and electrophoretic transfer of kidney proteins to nitrocellulose, Western blot will be performed as described by the vendor for the ECL plus kit (Amersham)for chemiluminescent detection. Bands on exposed films will be quantitated by densitometry. Nitrotyrosine from the vendor will be used as a positive control and GAPDH will be used as the loading control (10). Isoprostane (ISOP)- plasma and tissue: We will measure in plasma samples free levels of ISOP and in tissue samples total levels of ISOP using an enzyme immunoassay kit (EIA, Cayman). Basically all samples are thawed on ice prior to extraction, to reduce the likelihood of further production of ISOP. Tissue samples must first be homogenized in assay buffer with a polytron homogenizer, then undergo an alkaline hydrolysis with 15% potassium hydroxide to release the lipoprotein bound ISOP. Samples are then diluted with absolute ethanol (2X) and centrifuged to remove the precipitate. All samples are then diluted with distilled water, pHed to 3.0 with 1.0 HCI, and added to a Sep Pak C-18 extraction column (Milford, MA). Here they are washed first with distilled water and then Hexane, and eluted off the column with 99/1% Ethyl Acetate/Methanol. The organic solvent is evaporatedfrom the sample, which is reconstituted in assay buffer. For the assay, samples, tracer, and antiserum are added to wells pre-coated with mouse monoclonal secondary antibody and incubated overnight at room temperature. After incubation, plates are washed to remove all unbound ISOP and Ellman's Reagent (containing the substrate to acetylcholinesterase) is added to the wells. Absorbencies produced by this enzymatic reaction will be determined using a spectrophotometer set at 405nm and sample readings will be compared to the standards to obtain values (Fig.3) (4,8,11). Total Anti-oxidant Capacity - plasma This is afast andeasy assayfor measuring the combined actions of all antioxidants in biological fluids and there ability to reduce reactive oxygen species. The assay (AOP-490, Oxis Research) is based on the capacity of the antioxidants in the sample to reduce copper++ to copper +. Basically samples and standards are diluted 1:40 with Bathocuproine Reagent (combines with Copper + to form a compound that absorbs at 490 nm) then added to a microplate with baseline absorbance read at 490 nm. A copper ++ reagent is next added to the samples and standards and jncubated for 3 minutes. Final PHS 398/2590 (Rev.09/04, Reissued 4/2006) Page 316 Continuation Format Page Principal Investigator/Program Director (Romero, J.Carlos): Romero, J. Carlos COFG C absorbencies are read at 490mn and net absorbance is recorded. Sample absorbencies are then compared to those of standards with known quantities of uric acid to obtain Total Antioxidant Capacity for each sample. NADPH-oxidase (p47) - tissue NADPH (p47) is assayed using the western blot analysis. Briefly tissue samples are smashed at -80¿C and homogenized in lysis buffer. The homogenates are then centrifuged and supernatant removed. For the western blot, 50 ug sample supernatant (as determined by Lowry protein analysis) is loaded onto a 12.5 % Tris-HCI gel, then transferred to a PVDF membrane. Membranes are then blocked with 5 % milk in TBS-T and incubated overnight at 4¿C with primary p47-NADPH antibody (1:1000) (Cell Signalling). The next day membranes are incubated for 2 hours at room temperature with secondary (goat anti-rabbit) antibody (1:1000), fluoresced with ECL solution and intensities of the bands recorded on x-ray film. Finally membranes are stripped with guanidine hydrochloride solution, blocked with a 5% milk solution and incubated with B-actin antibody. Upon completion of all western analysis, intensities of p47 NADPH bands are quantified and values are computed relative to the intensities of B-actin(12). GSH/GSSG Assay - tissue (OXIS International. CA) Tissue samples are homogenized in lysis buffer added with 20 ul 1-Methy-2-vinyl-pyridium trifluoromethane sulfonate (M2VP), a thiol-scavenging reagent which rapidly scavenges GSH but does not interfere with glutathione reductase, since GSH oxidation likely occurs rapidly in disrupted tissues. Samples are vortexed with 5% metaphosphoric acid for 20 sec, and then added with assay buffer or GSSG buffer for final measurement. At the time of measurement, 200 pi of standards, blank or samples are added into the cuvettes, mixed with 200 ul Chromogen and 200ul glutathione reductase at room temperature for 5 min. 200 ul of NADPH is added to each cuvettes, the change of absorbance at 412nm recorded for 3 min. A six-point standard curve is constructed, and GSH or GSSG concentration, GSH/GSSG ratio was calculated following the instructions. DHE. In situ production of super oxide anion is measuredin 30-um frozen kidney sections using the oxidative fluorescent dye dihydroethidium (DHE). Cytosolic DHE exhibits blue fluorescence, but once oxidized by super oxide to ethidium bromide it intercalates within the cell's DNA, staining its nucleus a fluorescent red (excitation at 488 nm, emission 610 nm). Serial sections areequilibrated under identical conditions for 30 min at 37¿C in Krebs-HEPES buffer. Then, fresh buffer containing DHE (2 umol/l) is applied to each tissue section, cover slips added, and tissues incubatedfor 30 min in a light-protected humidified chamber at 37¿C. Quantification of each tissue section for levels of DHE is evaluated using fluorescence microscopy (13). Creatinine will be determined in plasma and urine by means of the Jaffe rate method. Sodium/Potassium will be measured in plasma and urine by flame photometry. StatisticalAnalysis Results will be shown as mean ¿ SEM. Comparisonswithin groups will be performed by use of paired Student's t test, and among groups by ANOVA, with the Bonferroni correction for multiple comparisons, followed by unpaired Student's t test. Literature cited 1. Schmitz C, Gottthardt M, Hinderlich S, Lehested J-R, Gross V, Vorum H, Christensen El, Luft FC, Takahashi S, Willnow TE. Normal Blood Pressure and Plasma Renin Activity in mice lacking the Renin- binding Protein, a Cellular Renin Inhibitor, J Biol Chem 275: 15357-15362, 2000. 2. Cheng H-F, Wang S-W, Zhang M-Z, McKanna JA, Breyer R, Harris RC. Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1. Am J Physiol 288:R638-R646, 2002. 3. Romero JC, Ruilope LM, Bentley MD, Fiksen-Olsen MJ, Lahera V, Vidal MJ . Comparison of the Effects of Calcium Antagonists and converting enzyme inhibitors on renal function under normal and hypertensive conditions. AM J Cardiol 62: 59G-68G, 1988. 4. Bolterman RJ, Manriquez MC, Ruiz MCO, Juncos LA, Romero JC. Effects of captopril on the renin angiotensin system, oxidative stress, and Endothelin in normal and hypertensive rats. Hypertension 46: 943-947, 2005. 5. Harding P, Carretero OA, Beierwaltes, WH. Chronic cyclooxygenase-2 inhibition blunts low sodium- stimulated renin without changing renal hemodynamics. J. Hypertension 18: 1107-1113, 2000. 6. Beierwaltes WH, Schryver S, Olson PS, Romero JC. Interaction of the prostaglandin and renin- angiotensin systems in isolated rat glomeruli. Am J Physiol 239: F602-F608, 1980 PHS 398/2590 (Rev.09/04, Reissued 4/2006) Page 317 Continuation Format Page

通过dia-sorin。该测定始于样品（零和孵化） 并在室温下与添加I125示踪剂的抗体涂层管中的标准孵育3小时。 去除熟料后，在伽马计数器和样品值中计数抗体涂层的管 通过将样品与标准曲线的样品进行比较而获得。然后减去零样品 从孵育的样品中确定生成的血管紧张素I的量（Figsl，2）（3,4）。在老鼠中，由于 PHS 398/2590（Rev.09/04，重新发行4/2006）Page 315延续格式页面 首席研究员/计划总监（Romero，J.Carlos）：Romero，J。Carlos Cotg c 测量PRA的问题，我们将衡量PRC。简短地用于中国一代，我们将使用 Carretero和Beierwaltes的方法（5,6）。这涉及在37»C中孵育2小时的小鼠样品2小时 在存在肽抑制剂的情况下，用肾切除型大鼠底物。为了终止反应，样品 将其煮沸10分钟，然后在2000 rpm时离心15分钟。然后将上清液取出，然后 存储在-20€c，直到使用与PRA测量相同的套件分配（DIA -SORIN）。 Anaiotensin II血浆和组织：要从组织中提取ANG II c，然后在冷蛋白中匀浆。然后将样品以20,000 g离心，Meoh上清液 将样品除去，冻干并存储在-80¿C上，直到重构为测定缓冲液进行提取。 重构的组织样品和等离子体样品将使用相同的方法提取和分析。 基本样品将在用DH2O洗涤并洗脱的预洗苯萃取柱上提取 和Meoh。通过冻干去除MEOH，并在测定缓冲液中重构样品。这 使用的酶免疫测定试剂盒由SPI-BIO提供，并由Cayman Chemical分布。它涉及 将样品和标准孵育与固定在96井板孔的单克隆抗体一起孵育。 免疫反应后，洗涤井，并将捕获的ANG II分子共价连接。 与戊二醛的抗体复合物。洗涤并定性后，绑定的ang II可以与 乙酰胆碱酶标记的抗体（示踪剂）。洗涤以去除未结合的示踪剂后，示踪剂绑定到 Ang II复合物与Ellman的试剂反应，以缓慢发展黄色 分光光度计板读取器的波长为405 nm。将样本的读数与 获得值的标准。该值作为组织样品的PG蛋白为PG，PG/ML的pg/ml 血浆样品。当测量20-100 pg时，该测定法显示2-7％的测定系数 当估计2-5 pg的ANG II时，ANG LL/ML和2-5％的变化（图2）（4,7,8,9）。 硝基伏苯菌 - 组织：该评估的原理是基于以下事实：NO是唯一的生物学 足够高的浓度产生的分子以超过溶剂的超氧化物形成〜OONO。这 化合物修饰蛋白质中的酪氨酸以产生硝基酪氨酸，使标记在体内可检测到标记。 然后，可以使用对硝基酪氨酸的单克隆抗体来确定免疫组织化学 依赖性酪氨酸硝化。在液氮中冷冻的肾脏组织将在20毫米HEPE中匀浆，pH 7.5，含有蛋白质抑制剂，将被离心600 xg以去除细胞碎片。凝胶 如前 将抗硝基酪氨酸单克隆抗体（Cayman，Ann Arbor）描述为主要抗体。 随后尺寸分离和肾脏蛋白向硝酸纤维素的电泳转移，Western印迹将 如供应商的ECL Plus Kit（Amersham）的化学发光检测所述进行。 暴露膜上的频带将通过光密度法定定量。供应商的硝基酪氨酸将被用作 阳性对照和GAPDH将用作加载控制（10）。 异丙烷（ISOP） - 血浆和组织：我们将在血浆样品中测量自由水平的ISOP和 在组织样品中，使用酶免疫测定试剂盒（EIA，Cayman）的ISOP总水平。基本上 在提取前将样品解冻在冰上，以减少进一步生产ISOP的可能性。组织 首先必须在测定缓冲液中使用多龙均匀均质均质样​​品，然后进行合金 用15％氢氧化钾水解释放脂蛋白结合的ISOP。然后将样品稀释 与绝对乙醇（2x）一起离心以去除沉淀物。然后将所有样品稀释 蒸馏水，用1.0 HCI phe至3.0，并添加到Sep Pak C-18提取柱（马萨诸塞州米尔福德）中。 在这里，它们首先用蒸馏水和己烷洗涤，然后用99/1％的乙基洗脱。 乙酸/甲醇。从样品中蒸发有机溶液，该样品是在测定中重构的 缓冲。对于测定，将样品，示踪剂和抗血清添加到用小鼠单克隆的预涂层中 二级抗体并在室温下孵育过夜。孵育后，将板洗净到 添加所有未结合的ISOP和Ellman的试剂（含有乙酰胆碱酯酶的底物） 到井。该酶促反应产生的吸收剂将使用A确定 将分光光度计设置为405nm，并将样品读数与获得值进行比较 （图3）（4,8,11）。 总抗氧化能力 - 等离子体这是用于测量合并作用的AFAST和EASY测定 在生物液中的所有抗氧化剂和减少活性氧的能力。测定（AOP-490， OXIS研究）基于样品中抗氧化剂的能力，将铜++降低为铜+。 基本的样品和标准用巴达科蛋白试剂稀释1:40（与铜 +结合到 形成一种在490 nm处吸收的化合物，然后将基线吸光度读取为490 NM。接下来将铜++试剂添加到样品和标准品中，并进行3分钟。最终的 PHS 398/2590（Rev.09/04，重新发行4/2006）第316页延续格式页面 首席研究员/计划总监（Romero，J。Carlos）：Romero，J。CarlosCofg C 在490mn的吸收率上，记录了净吸光度。然后将样品吸收与 具有已知量尿酸的标准品以获得每个样品的总抗氧化能力。 NADPH-氧化酶（P47） - 使用蛋白质印迹分析分配组织NADPH（P47）。简要地 将组织样品粉碎在-80¿C处，并在裂解缓冲液中匀浆。那时是匀浆 离心和上清液已移除。对于Western blot，50 ug样品上清液（由 Lowry蛋白分析）将其加载到12.5％的Tris-HCI凝胶上，然后转移到PVDF膜上。 然后将膜用TBS-T中的5％牛奶阻塞，并在4°C下与主要P47-NADPH孵育过夜 抗体（1：1000）（细胞信号传导）。第二天将膜在室温下孵育2小时 用二次（山羊抗兔）抗体（1：1000），用ECL溶液和带的强度荧光 录制在X射线胶片上。最后，膜被盐酸盐溶液剥离，用A阻塞 5％牛奶溶液并与B-肌动蛋白抗体孵育。完成所有西方分析后， 量化P47 NADPH频段，并计算值相对于B-肌动蛋白的强度（12）。 GSH/GSSG测定 - 组织（OxisInternational。CA）组织样品在裂解缓冲液中匀浆 添加20个UL 1-甲基-2-乙烯基 - 吡啶甲烷磺酸盐（M2VP），一种硫醇扫描试剂 它迅速清除GSH，但不会干扰谷胱甘肽的减少，因为GSH氧化可能很可能 发生在破坏的组织中。样品用5％的互磷酸涡旋20秒，然后 添加了测定缓冲液或GSSG缓冲液以进行最终测量。在测量时，200 pi 标准，空白或样品被添加到比色杯中，与200 ul Chromogen和200 ul谷胱甘肽混合 在室温下降低5分钟。每个比色杯添加了200 ul nADPH，变化 412nm的吸光度记录了3分钟。构建了六点标准曲线，GSH或GSSG 按照说明计算浓度，GSH/GSSG比率。 dhe。超级氧化物阴离子的原位生产是使用30-UM冷冻肾脏的测量 氧化荧光染料二氢（DHE）。胞质DHE表现出蓝色荧光，但一旦被氧化 通过超级氧化物到溴化乙锭，它在细胞的DNA中插入，染色的核呈荧光红色 （488 nm的激发，发射610 nm）。在相同条件下进行30分钟的连续切片进行平衡 在克雷布斯 - 螺旋缓冲液中的37°C。然后，将含有DHE（2 Umol/L）的新鲜缓冲液应用于每个组织截面， 盖上盖载玻片，并在37°C的一个光保护的加湿腔室中孵育30分钟。 使用荧光显微镜评估每个组织段的DHE水平（13）。 肌酐将通过jaffe速率方法在血浆和尿液中确定。 钠/钾将通过火焰光度法在血浆和尿液中测量。 统计分析 结果将显示为平均值�SEM。将使用配对进行比较组的比较组 学生的T测试以及ANOVA组中的小组，并进行了多次比较的Bonferroni校正， 其次是未配对的学生的T测试。 引用文献 1。SchmitzC，Gottthardt M，Hinderlich S，Lehested J-R，Gross V，Vorum H，Christensen EL，Luft FC， 高桥S，威尔诺。缺乏肾素的小鼠中血压和血浆肾素活性 结合蛋白，一种细胞肾素抑制剂，J Biol Chem 275：15357-15362，2000。 2。ChengH-F，Wang S-W，Zhang M-Z，McKanna JA，Breyer R，Harris RC。前列腺素会增加 响应ACE抑制的肾素产生不是从环氧酶-1中得出的。 Am J Physiol 288：R638-R646，2002。 3。RomeroJC，Ruilope LM，Bentley MD，Fiksen-Olsen MJ，Lahera V，Vidal MJ。效果的比较 钙拮抗剂和在正常和 高血压条件。 Am J Cardiol 62：59G-68G，1988年。 4。BoltermanRJ，Manriquez MC，Ruiz MCO，Juncos LA，Romero JC。卡托普利对肾素的影响 血管紧张素系统，正常和高血压大鼠中的血管紧张素系统，氧化应激和内皮素。高血压46： 943-947，2005。 5。HardingP，Carretero OA，Beierwaltes，WH。慢性环氧合酶-2抑制作用钝性低钠 刺激肾素而不改变肾脏血流动力学。 J.高血压18：1107-1113，2000。 6。BeierwaltesWH，Schryver S，Olson PS，Romero JC。前列腺素和肾素的相互作用 - 分离的大鼠肾小球中的血管紧张素系统。 Am J Physiol 239：F602-F608，1980 PHS 398/2590（Rev.09/04，重新发行4/2006）第317页延续格式页面