Tyrosine Hydroxylase Activity in Rat Brain Synaptosomes: Direct Measurement Using High Performance Liquid Chromatography

Abstract: By use of high performance liquid chromatography with electrochemical detection to measure dopamine production, tyrosine hydroxylase (EC 1.14.16.2) activity has been measured in rat brain synaptosomes from striatum and forebrain. Normal specific activities three- to fivefold higher than previously reported in the literature for radiochemical methods of assay were found. It is suggested that synaptosomes contain a significant amount of endogenous substrate for tyrosine hydroxylase, which causes dilution of the added labelled tyrosine and hence underestimation of the activity of this enzyme when radiochemical methods are used.     

A Rapid and Simple Method for the Determination of Picogram Levels of 3-Methoxytyramine in Brain Tissue Using Liquid Chromatography with Electrochemical Detection

Abstract: A rapid and simple technique using solvent extraction, ion-pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3-methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, ±4%); recovery of authentic 3-methoxytyramine added to the samples is 45–50%. 3-Methoxytyramine levels found with this technique in rat striata were 15 ± 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3-methoxytyramine levels in rat striata, while catechol- O -methyltransferase inhibition with 3',4'-dihydroxy-2 methylpropiophenone completely depleted 3-methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D-amphetamine were also examined. The 3-methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.     

常见肠道菌群代谢产物作为疾病诊断的指针的研究进展

人类肠道菌群能够产生多种代谢产物或与人体相互作用产生肠道菌群-宿主共代谢物,显著影响人体各大系统的生理功能。当人体健康状态以及肠道菌群发生变化时,肠道代谢物的种类和含量也会相应受到影响,因此肠道菌群代谢产物具有作为疾病诊断指针的巨大潜力。本文总结了常见的几类肠道微生物代谢产物,包括糖类、胆碱代谢物、脂质、氨基酸与肽类、维生素、胆汁酸、短链脂肪酸、酚、苯甲酰基和苯基衍生物等,及其在不同疾病状态下的作用机理,以期更好地理解肠道菌群、代谢产物和疾病之间的相关性,为疾病的预防、诊断和治疗提供新的靶点。     

4种染色方法对甜瓜白粉病菌染色效果的观察比较

考马斯亮蓝组织透明染色方法可以清楚观察到白粉病菌的5个发育阶段,即萌发的分生孢子、初生芽管、胞间菌丝、分生孢子梗和后期菌落。考马斯亮蓝几乎使寄主组织不着色,不产生背景色干扰,而菌体变深蓝色。苯胺蓝组织透明染色方法也可观察到病菌的不同发育阶段,但苯胺蓝易使寄主组织产生浅蓝色背景,而菌体呈现深蓝色,观察效果不理想。荧光素钠和苯胺蓝两种荧光染色方法均能使菌体在紫外或者蓝光下产生黄绿色荧光,而寄主组织呈现黑色背景,强烈的反差利于观察。荧光素钠可以观察到菌体整个发育阶段。苯胺蓝只适合于前期菌体入侵过程的观察。     

A new family of CRISPR‐type V nucleases with C‐rich PAM recognition

Most CRISPR‐type V nucleases are stimulated to cleave double‐stranded (ds) DNA targets by a T‐rich PAM, which restricts their targeting range. Here, we identify and characterize a new family of type V RNA‐guided nuclease, Cas12l, that exclusively recognizes a C‐rich (5''‐CCY‐3′) PAM. The organization of genes within its CRISPR locus is similar to type II‐B CRISPR‐Cas9 systems, but both sequence analysis and functional studies establish it as a new family of type V effector. Biochemical experiments show that Cas12l nucleases function optimally between 37 and 52°C, depending on the ortholog, and preferentially cut supercoiled DNA. Like other type V nucleases, it exhibits collateral nonspecific ssDNA and ssRNA cleavage activity that is triggered by ssDNA or dsDNA target recognition. Finally, we show that one family member, Asp2Cas12l, functions in a heterologous cellular environment, altogether, suggesting that this new group of CRISPR‐associated nucleases may be harnessed as genome editing reagents.     

植物原生质体在分子细胞生物学研究中的应用

植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。     

基于CRISPR/Cas系统的生物传感策略研究进展

CRISPR/Cas9系统是继锌指核酸内切酶、类转录激活因子效应物核酸酶之后的第三代基因组定点编辑工具,因其具有特异性切割双链DNA的能力,被广泛应用于基因编辑、生物传感等领域。Cas12a(Cpf1)、Cas13a(C2c2)等蛋白"附属切割"活性的发现,拓展了CRISPR/Cas系统在生物传感中的应用。近年来,研究人员开发出一系列快速、超敏、高特异性的生物传感系统用于分子检测,如SHERLOCK,DETECTR等。本文主要综述了基于CRISPR/Cas系统的生物传感策略的研究进展,并展望了其未来发展的方向。     

Direct competitive chemiluminescence immunoassays based on gold‐coated magnetic particles for detection of chloramphenicol

Direct competitive chemiluminescence immunoassays (CLIA) based on gold‐coated magnetic nanospheres (Au‐MNPs) were developed for rapid analysis of chloramphenicol (CAP). The Au‐MNPs were modified with carboxyl groups and amino groups by 11‐mercaptoundecanoic acid (MUA) and cysteamine respectively, and then were respectively conjugated with CAP base and CAP succinate via an activating reaction using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS). NSP‐DMAE‐NHS, a new and effective luminescence reagent, was employed to label anti‐CAP antibody (mAb) as a tracer in direct CLIA for CAP detection using a ‘homemade’ luminescent measurement system that was set up with a photomultiplier tube (PMT) and a photon counting unit linked to a computer. The sensitivities and limits of detection (LODs) of the two methods were obtained and compared according to the inhibition curves. The 50% inhibition concentration (IC₅₀) values of the two methods were about 0.044 ng/mL and 0.072 ng/mL respectively and LODs were approximately 0.001 ng/mL and 0.006 ng/mL respectively. To our knowledge, they were much more sensitive than any traditional enzyme‐linked immunosorbent assay (ELISA) ever reported. Moreover, the new luminescence reagent NSP‐DMAE‐NHS is much more sensitive and stable than luminol and its derivatives, contributing to the sensitivity enhancement. Copyright © 2015 John Wiley & Sons, Ltd.