Comparison of the photochemical behavior of four different photoactivatable probes

The most commonly used photoaffinity labeling probes are compared, which are aryl azides, aryl diazirines, α-diazocarbonyls and benzophenone-derivatives. The compounds were used under identical conditions and crosslinking efficiency, influence of water, irradiation requirements, and by-products were investigated. Using the pentapeptide thymopentin (TP5) as a model system, we synthesized four analogues by solid-phase peptide synthesis and partially N-terminal modification to obtain [p-(3-trifluoromethyl)diazirinophenylalanine⁵] TP5, [p-benzoylphenylalanine⁵] TP5, 4-azidobenzoyl-TP5 and 2-diazo-3, 3, 3-trifluoropropionyl-TP5. The peptides were characterized by HPLC and ion-spray mass spectroscopy. Irradiation of the peptides with two different ultraviolet sources was carried out in water, n-propanol and water/n-propanol to imitate both hydrophobic and hydrophilic peptide/protein-interactions as well as the influence of the aqueous environment. Analysis of the products with HPLC, ion-spray MS, HPLC-MS and HPLC-CID-MS revealed that (Tmd)Phe is a highly potent carbene-precursor, which can be transformed easily into uniform crosslinking products by smooth photolysis. However, the electrophilic nature of the intermediate causes a high tendency to react with water molecules. The 4-azidobenzoyl group showed comparable crosslinking efficiency, but the probability to create non-uniform irradiation products (e.g. through rearrangement) is higher, whereas the reaction with water is less dominant. In contrast, Bpa was found to have an extremely low affinity to react with water, whereas prolonged UV irradiation is needed to get complete rearrangement into a variety of products. As the absorption band of α-diazocarbonyls at around 350 nm possesses a low extinction coefficient, 2-diazo-3, 3, 3-trifluoropropionyl-TP5 could not be activated at all with the optimized irradiation conditions that we have chosen for our comparative studies. © Munksgaard 1997.     

Effects of P-Aminophenyl Dichloroarsine on Reduced High-affinity [3H]Nicotine Binding Sites from Chick Brain: A Covalent,Yet Reversible,Agent for Neuronal Nicotinic Receptors

Neuronal nicotinic acetylcholine receptor (nAChR) α-subunits contain a conserved disulphide that is essential for function. Here, we have examined the effects of sulphydryl redox reagents on [³H]nicotine binding to chick brain nAChR immunoisolated with the monoclonal antibody mAb35. The disulphide reducing agent, dithiothreitol (DTT), inhibited [³H]nicotine binding [50% inhibitory concentration (IC₅₀) = 146 μM] but this effect was reversed (93±1.5%) by subsequent reoxidation with 1 mM dithio-bis(nitrobenzoic acid) (DTNB). The trivalent arsenical, p -aminophenyl dichloroarsine (APA), which reacts with pairs of spatially close sulphydryls, was a potent inhibitor of reoxidation by DTNB (IC₅₀= 35 nM). However, application of the 'anti-arsenical', 2,3-dimercaptopropane sulphonic acid (DMPS), restored agonist binding after APA treatment (50% effective concentration = 120 μM). Paradoxically, DMPS was also found to be a potent oxidizing agent of these receptors. Affinity alkylation of reduced nAChRs with bromoacetylcholine (BAC; 100 μM) irreversibly blocked nicotine binding (>90%). We propose (but have not proven) that APA interacts with the cysteines homologous to Cys192–193 in Torpedo AChRs, since APA pretreatment of reduced neuronal receptors protected against irreversible BAC alkylation, as shown by subsequent reversal of DMPS (2 mM; 20 min). This study illustrates the potent and reversible nature of the arsenical's covalent interaction with an isolated nAChR and suggests that modified arsenicals could be useful nAChR probes.     

The potential of DBP gels containing intervertebral disc cells for annulus fibrosus supplementation:in vivo

Demineralized bone particle (DBP), which is widely used as a biomaterial in the field of tissue engineering, contains various bioactive molecules, such as cytokines. For this reason, in this study we investigated the effects of injectable DBP gels on cell proliferation, inflammation and maintenance of the shape of DBP gels as a scaffold able to substitute for intervertebral discs (IVDs) in vivo. DBP gels were fabricated with different percentages (5% and 10%) of DBP powder and 3% acetic acid, including 0.02% pepsin. DBP gels with 1 × 10⁶ annulus fibrosus (AF) cells were implanted into the dorsal subcutaneous region of BALB/C‐nu mice for 1, 2 and 3 weeks. Cell proliferation was measured by MTT assay. The effect of DBP gels on the inflammatory response was analysed by measuring the amount of tumour necrosis factor‐alpha (TNFα) released. Also, histological methods were carried out to analyse the response of DBP gels in vivo. This study demonstrated that injectable DBP gels are able to provide physical scaffolds for growing IVD cells in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

不同戊型肝炎病毒IgG抗体检测试剂盒临床对比分析

目的比较磁微粒化学发光试剂与酶联免疫吸咐测定试剂检测戊型肝炎病毒Ig G抗体的结果差异。方法选取企业样本库2010年1月至2012年5月住院和门诊患者935例,分别用A厂家酶联免疫吸咐测定试剂和郑州安图生物磁微粒化学发光试剂测定戊型肝炎病毒Ig G抗体平行检测,不符样本用C厂家酶联免疫吸咐测定试剂复测。结果郑州安图生物磁微粒化学发光试剂和戊型肝炎病毒Ig G抗体A厂家酶联免疫吸咐测定试剂阳性一致百分比为95.5%,阴性一致百分比为97.7%,总符合率为97.2%,差异无统计学意义(P>0.05)。结论磁微粒化学发光戊型肝炎病毒Ig G抗体试剂与酶联免疫吸咐测定试剂临床性能相似,适于临床推广应用。     

人类基因检测相关试剂评价国家参考品研制技术要求介绍

目的：进一步规范人类基因检测相关试剂评价国家参考品的研制工作。方法：人类基因检测相关试剂评价国家参考品是指用于人类基因检测相关试剂质量评价的参考物质。本文详细介绍了这类参考物质的原材料筛选、制备、标定、稳定性研究、包装、储存及供应方面的要求。结果与结论：人类基因检测相关试剂评价国家参考品研制过程更加清晰,管理更加规范。     

转染试剂及幽门螺杆菌对AGS细胞形态的影响

目的:探讨不同的转染试剂对AGS细胞形态的影响,观察这些转染试剂能否像幽门螺杆菌那样引起AGS细胞蜂鸟状改变．方法:用多聚阳离子转染试剂(多聚乙酰亚胺、梭华-Sofast)和改良的脂类转染试剂 (Effectene Transfection Reagent)分别转染AGS 细胞,观察细胞形态的变化．用幽门螺杆菌 26695菌株攻击AGS细胞,观察细胞形态的变化并与经转染试剂作用的细胞进行形态比较．结果:幽门螺杆菌攻击后4 h能引起AGS细胞发生蜂鸟状改变．两种多聚阳离子转染试剂也均能引起AGS细胞发生蜂鸟状改变,与幽门螺杆菌引起的细胞形态改变相似,大部分细胞拉长,并且不受是否转染DNA的影响．此种变化在转染4 h时便出现,与试剂剂量的增加呈正相关．当PEI的剂量为10 μL时,细胞出现凋亡,15μL时凋亡加重．改良的脂类转染试剂对 AGS细胞的形态未产生影响．结论:在研究引起AGS细胞形态改变的因素时,不宜选择多聚阳离子转染试剂;多聚阳离子引起的AGS细胞形态改变与幽门螺杆菌引起的AGS细胞形态改变之间是否存在关系值得进一步研究．     

化学法交联人-人抗体用于抗甲型肝炎病毒IgM抗体检测质控品的研究

生部临床检验中心发放的抗-HAV IgM质控品相当,S/CO值分别为2.96、3.12,能够作为抗-HAV IgM检测的阳性质控.该嵌合抗体在4℃及-20℃条件下均能稳定保存至少6周.IgG、IgM及交联剂不影响嵌合抗体的阳性检测结果,且该嵌合抗体与抗-HBc IgM、抗-HEV IgM检测不存在交叉反应.使用临床上常见的检测试剂盒检测结果均为阴性.结论 通过化学交联的方法成功构建了人-人嵌合抗体,可用作为抗-HAV IgM检测质控物.     

COULTER流式细胞仪采用自配试剂在CD55、CD59水平检测中的应用探讨

目的探讨美国COULTER流式细胞仪试剂国产化问题。方法将自配鞘液及清洗液的理化性质、精密度、准确度、稳定性与原装试剂进行对比分析。结果自配试剂与原装试剂理化性质一致,采用自配试剂测定15份患者标本CD55、CD59水平并与原装试剂对比,差异无统计学意义（P〉0．05）,两者呈高度正相关（r〉0．95）。自配试剂稳定性好,放置1年后,用原装试剂分别测定15份标本,结果差异无统计学意义（P〉0．05）,两者呈高度正相关（r〉0．95）。结论自配美国COULTER流式细胞仪试剂完全可以代替原装进口试剂进行CD55、CD59的检测。