A Biotin–Avidin-Based Enzyme Immunoassay for βh-Endorphin

An avidin–biotin enzyme-linked immunosorbent assay (ELISA) is described for _h-endorphin (_h-EP). Microtiter plates coated with commercially available antibodies were used together with _h-EP tracer derivatives that were biotinylated in positions 24, 28, and 29 via a C₆ spacer arm. Nonspecific binding of biotinylated derivatives to the microtiter plates was blocked with a mixture of 1% casein and 10% ethanolamine in 0.1 M NaHCO₃. A sequential saturation procedure using a high-affinity antiserum in combination with an avidin–alkaline phosphatase complex matched the sensitivity of reported radioimmunoassays (RIAs), with a detection limit of 0.5 fmol/assay. The intra- and interassay coefficients of variation were 5 and 12%, respectively. Results obtained by ELISA and RIA showed good correlations (r = 0.95). The -EP concentration in extracted rat plasma after high-performance liquid chromatographic (HPLC) fractionation was determined by this method to be 1600 fmol/ml.     

Cotinine determination by immunoassays may be influenced by other nicotine metabolites

Polyclonal rabbit anticotinine antiserum, which can be used for biomonitoring nicotine uptake by the determination of cotinine in body fluids, was checked by a competitive ELISA for its cross-reactivity with nine nicotine metabolites. The highest percentage of relative crossreactivity (about 30%) was observed with trans-3-hydroxycotinine, a metabolite which is known to be excreted in 3-fold higher amounts than cotinine in the urine of human smokers. Therefore, it is possible that cotinine determinations performed by immunochemical methods — especially in urine — may yield overestimated cotinine concentrations.     

Deacetylation and further metabolism of the mercapturic acid of hexachloro-1,3-butadiene by rat kidney cytosol in vitro

Hexachloro-1,3-butadiene (HCBD) is more nephrotoxic to female than male rats. Metabolism of HCBD involves conjugation with glutathione followed by formation of the cysteine conjugate S-(pentachloro-1,3-butadienyl) cysteine (PCBD-CYS) and then the mercapturic acid N-acetyl-S-pentachloro-1,3-butadienyl-cysteine (PCBD-NAC). PCBD-NAC is also more nephrotoxic to female rats than male rats. The deacetylation of [¹⁴C]-PCBD-NAC to PCBD-CYS and the binding of radiolabelled metabolites to protein has been studied using renal cytosol preparations from male and female rats in vitro, since a sex-related difference in these reactions could explain the difference in nephrotoxicity found in vivo. PCBD-NAC was rapidly metabolised by renal cytosol. The rate of metabolism was similar with either male or female renal cytosol, and the major metabolite identified was PCBD-CYS. N-Acetylation of PCBD-CYS to PCBD-NAC was not detected in the presence of either male or female renal cytosol. Covalent binding of radioactivity from [¹⁴C]-PCBD-NAC to cytosolic protein could be detected after 5 min incubation, and although the extent of binding was similar for both male and female cytosol at early time periods, after 60 min incubation more binding was found in the presence of male cytosol. Covalent binding was largely prevented by aminooxyacetic acid, an inhibitor of cysteine conjugate -lyase, suggesting a role for this enzyme in the activation of HCBD. These results indicate that the sex differences in the nephrotoxicity of HCBD and PCBD-NAC in the rat are not attributable to differences in the rate of deacetylation of PCBD-NAC to give the proximate nephrotoxin PCBD-CYS.This work was supported by a fellowship from the European Science Foundation granted to I. S. P.     

Induction of light hydrocarbon nephropathy by p-dichlorobenzene

In order to clarify the etiology of a dose-related increase in the incidence of tubular cell adenocarcinomas of the kidney in male rats, the nephrotoxicity of p-dichlorobenzene (p-DCB) was investigated in a subchronic study. Groups of ten male and ten female Fischer 344 rats were dosed by gavage with 0 (controls), 75, 150, 300 or 600 mg p-DCB/kg/day in corn oil. Half of the animals were sacrificed after 4 weeks and the remainder after 13 weeks. Increased urinary LDH and epithelial cell excretion and exacerbation of hyaline droplet accumulation in the cytoplasm of renal cortical cells were observed in male rats over the entire dose range investigated. Tubular single cell necrosis, dilated tubules with granular cast formation in the outer zone of the medulla, were evident in male rats after 4 and 13 weeks of treatment with doses of 150–600 mg/kg/day. In female rats there was no indication of a nephrotoxic action of p-DCB. The effects on the kidney, both in their morphological characteristics and the fact that they occur exclusively in male animals, correspond to the light hydrocarbon nephropathy observed as a result of short-term treatment with a number of aliphatic and cyclic hydrocarbons. The development of cortical renal tumors seems to be associated with this kind of kidney damage which is unique to male rats. The literature on this subject generally regards these renal effects as not predictive for man.     

Thiol Reduction of 3′-Azidothymidine to 3′-Aminothymidine: Kinetics and Biomedical Implications

The ability of thiols to reduce 3-azidothymidine (AZT) to 3-aminothymidine has been investigated. Incubation with glutathione, dithiothreitol (DTT), or mercaptoethanol at pH 7.2 and 37°C leads to quantitative reduction of the azido moiety to an amine. The reaction is first order in AZT and first order in reducing agent (mono- or dithiol). The second-order rate constants are 2.77 × 10^–3, 6.55 × 10^–5, and 6.35 × 10^–6 M ^–l sec^–1 for the dithiothreitol, glutathione, and mercaptoethanol reductions, respectively. The thiol reduction of alkyl azide to amine under mild conditions is a synthetic method particularly suitable for water-soluble azido compounds that are sensitive to catalytic hydrogenation. The potential for the mono- or dithiol-mediated reduction of alkyl azides under biological conditions must be considered when conducting studies of azido drugs.     

The Relationship of pK a and Acute Skin Irritation in Man

The relationship between pK _a and skin irritation in man is studied for a homologous series of benzoic acid derivatives, which permeate through human skin at comparable rates (15–88 µg/cm²/hr). Skin irritation and pK _a are correlated for pK _a 4. Laser Doppler velocimetric assessment of skin blood flow, color meter readings, erythema, edema, and the primary irritation index are all linearly correlated and related to pK _a, erythema at 24 hr appears to be the most sensitive parameter to variation in pK _a when pK _a 4.     

踝肱指数和肱踝动脉脉搏波速度与血管舒张功能联合载脂蛋白B与载脂蛋白A-1比值对中青年冠心病血瘀证的预测价值研究

背景 冠心病属中医“胸痹心痛”范畴,其证候分布以血瘀证最为多见,与老年人相比,中青年血瘀证的发生率更高。本研究在团队前期研究基础上进行拓展,首次运用动脉弹性和血管内皮功能检测联合载脂蛋白比值诊断中青年冠心病血瘀证,旨在为早期发现中青年冠心病血瘀证提供新思路,也可为优化补充冠心病血瘀证的诊断标准提供参考。目的 探讨踝肱指数(ABI)、肱踝动脉脉搏波速度(baPWV)、血管舒张功能(FMD)联合载脂蛋白B与载脂蛋白A-1比值(apoB/apoA-1)与中青年冠心病血瘀证的关系及其预测价值。方法 选取2016年12月至2021年12月于中日友好医院中西医结合心脏内科住院治疗的中青年(<50岁)冠心病患者,并根据《冠心病血瘀证诊断标准》分为血瘀证组和非血瘀证组。收集患者首次冠状动脉造影前的ABI、baPWV、FMD和apoB/apoA-1等临床资料。采用多因素Logistic回归分析构建中青年冠心病患者血瘀证的预测模型,并采用受试者工作特征(ROC)曲线进行预测价值评价。结果 本研究共纳入中青年冠心病患者206例,其中血瘀证组127例,非血瘀证组79例。多因素Logistic回归分析结果...     

转录因子Ascl2调控Acss1/3的表达影响结直肠癌患者预后

转录因子Ascl2作为WNT信号靶基因,可影响结直肠癌（CRC）前体细胞干性特征,探讨其能否调控短链脂肪酸β-氧化而影响CRC患者的预后。方法 下载稳定干扰Ascl2表达的CRC LS174T细胞（sh-Ascl2/LS174T）的mRNA及miRNA差异表达数据（GSE69036和GSE34926）分析其靶基因;应用RT-PCR方法和Western 印迹定量检测sh-Ascl2/LS174T及对照细胞中的Ascl2、Acss1和Acss3的mRNA表达水平,以及Ascl2和Acss1蛋白表达水平,从GSE44076表达数据和UALCAN数据比较在正常结直肠粘膜、CRC组织和不同临床病理分期的肿瘤组织的表达水平差异;TCGA数据库下载548例CRC患者基因 mRNA表达量及相关临床信息,用Spearman等级相关分析表达相关性,Kaplan-Meier法进行生存分析,Cox回归分析评估危险因素。结果 sh-Ascl2/LS174T细胞中Acss1的mRNA较对照细胞下调2.08倍,miR-4282表达水平下调2.069倍。RT-PCR定量检测sh-Ascl2/LS174T细胞中Acss1的mRNA和miR-4282水平明显下降(P＜0.01), Acss3的mRNA水平明显升高(P＜0.05);Acss1和Ascl2蛋白水平较对照细胞均明显下降。CRC组织Ascl2和Acss1的mRNA水平明显高于癌旁结直肠粘膜组织(P＜0.001),而Acss3明显低于癌旁结直肠粘膜组织 (P＜0.001);CRC组织中Ascl2与Acss1的mRNA表达水平呈正相关,与Acss3的表达水平呈负相关(均P＜0.001)。Ascl2和Acss3表达水平与疾病特异性生存期（DSS）、总体生存期（OS）、无进展生存期（PFS）无关,Acss1高表达组比低表达组的DSS (P=0.0389)和OS (P=0.04)明显降低。Ascl2与Acss1基因异常表达、Ascl2、Acss1和Acss3基因异常联合表达与CRC患者的DSS存在显著的联系(P=0.0377和P=0.0161),Ascl2、Acss1和Acss3三个基因的联合异常表达是影响CRC患者DSS的危险因素之一(P=0.043)。结论Ascl2对CRC细胞中的Acss1/3的表达可能具有调控作用而导致其短链脂肪酸β-氧化的重编程,它们的联合异常表达可以一定程度预测CRC患者的预后     

右美托咪定调控Nrf2/HO-1通路对H2O2诱导心肌细胞氧化应激损伤的作用研究

目的 探讨右美托咪定调控核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)通路对过氧化氢(H₂O₂)诱导心肌细胞氧化应激损伤的作用。方法 体外培养大鼠H9C2心肌细胞,设置对照组、H₂O₂组、1μmol右美托咪定+H₂O₂组、5μmol右美托咪定+H₂O₂组、10μmol右美托咪定+H₂O₂组。CCK-8法检测各组H9C2细胞增殖情况;酶联免疫吸附试验(ELISA)检测各组H9C2细胞丙二醛(MDA)、超氧化物歧化酶(SOD)水平;实时荧光定量聚合酶链反应(q RT-PCR)检测各组H9C2细胞Nrf2、HO-1 mRNA相对表达量;Western blotting检测各组H9C2细胞Nrf2、HO-1蛋白相对表达量。结果 与对照组比较,H₂O₂组H9C2细胞存活率、SOD水平、Nrf2、HO-1 mRNA及蛋白相对...     

格隆溴铵对高氧诱导幼鼠急性肺损伤的影响及其机制研究

目的 探究格隆溴铵对高氧诱导幼鼠急性肺损伤（ALI）的影响及作用机制。方法 从30只SD幼鼠中随机选取10只为对照组,其余幼鼠成功复制高氧诱导的ALI模型,随机分为ALI组、格隆溴铵组,每组10只。格隆溴铵组雾化吸入0.8 mg/（kg·d）格隆溴铵,ALI组、对照组吸入等体积生理盐水,连续给药7 d后,测量幼鼠肺组织湿/干重比值（W/D）、肺指数,检测白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）及肿瘤坏死因子-α（TNF-α）水平及血清活性氧基团（ROS）、超氧化物歧化酶（SOD）水平,比较肺组织病理学变化及Toll样受体4/髓分化因子88（TLR4/MyD88）通路蛋白的表达。结果 与对照组比较,ALI组W/D及肺指数升高（P <0.05）,血清IL-1β、IL-6、TNF-α、SOD水平升高（P <0.05）,ROS水平降低（P <0.05）,TLR4、MyD88蛋白相对表达量上调（P <0.05）;与ALI组比较,格隆溴铵组W/D及肺指数降低（P <0.05）,血清IL-1β、IL-6、TNF-α、SOD水平降低（P <0.05）,ROS水平升高（P <0.05）,TLR4、MyD88蛋白相对表达量下调（P <0.05）。结论 格隆溴铵能改善血清炎症指标及氧化应激指标,降低高氧诱导的ALI,其作用机制可能与TLR4/MyD88通路有关。