一种改进的沙蒿总RNA的提取方法

介绍了一种适用于沙蒿总RNA提取的方法。此方法有效地克服了沙蒿中富含的多糖类物质———沙蒿胶对总RNA提取的干扰。运用此方法所提取的RNA纯度高 ,完整性较好 ,可满足多数分子生物学实验的需要。     

重组铜绿假单胞菌外毒素结构域Ia片段的佐剂功效研究

采用分子克隆技术,将铜绿假单胞菌PA103株编码的外毒素结构域Ia(Domain Ia)的基因重组于原核表达载体pET-42b( )上,构建了pET-EPA103蛋白表达载体。转化感受态大肠杆菌DE3。经IPTG诱导表达,初步纯化表达蛋白,用以免疫BALB／c纯系小鼠。制备小鼠脾淋巴细胞悬液,经刀豆素A(ConA)刺激后,用MTT比色法检测特异性淋巴细胞增殖反应。通过rEPA皮下注射BALB／c小鼠耳廓,诱导小鼠迟发型过敏反应(DTH)。采用特异性淋巴细胞增殖反应和DTH试验来检测pET-EPA103表达蛋白所引起的小鼠细胞免疫应答水平,淋巴细胞增殖情况与DTH均可间接反映细胞免疫应答水平,进而评价重组铜绿假单胞菌外毒素(rEPA)Domain Ia蛋白片段的佐剂功效。     

股骨头负重区松质骨的压缩力学特性

本文对19具新鲜青年(30左右)尸体股骨负重区的松质骨进行了压缩测试,测算并提供了一组松质骨弹性模量,极限应力,应变,屈服载荷及能量吸收值。试验表明松质骨力学性能主要缓解外力冲击加压。     

Evaluation of Colilert-18 for Detection of Coliforms and Eschericha coli in Subtropical Freshwater

The accuracy of Colilert-18 as a test for coliforms and Escherichia coli in subtropical freshwater was evaluated by using API 20E strips and fatty acid methyl ester analysis. The false-positive and -negative rates of detection were 7.4 and 3.5%, respectively, for E. coli and 9.6 and 6.3%, respectively, for coliforms.     

二龄草鱼脾脏、肝脏组织高表达甘露糖结合凝集素mRNA

Innate immunity is expected to be very important in fish. Mannose-bingding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. In this experiment, total mRNA was isolated from spleen, liver, gills, thymus, head kidney and kidney of adult and immature grass carp Ctenopharygodon idllus. The cDNA of MBL was obtained by RT-PCR using total mRNA from the spleen of carp as template. Such cDNA was labled with ^32p and used as probe for Northern analysis, and autoradiographic signals were quantified by densitometry analysis. The results showed that MBL was high expressed in the spleen and liver and low in gills, thymus, head kidney and kidney of adult grass carp, and MBL was much lower expressed in spleen and liver of immature grass carp than those of adult grass carp. The results might partially explain why immature grass carp are vulnerable to grass carp hemorrhage virus (GCHV) whereas adult grass carp are not.This suggested that MBL mav be an imoortant anti-GCHV factor [Acta Zoologica Sinica 50 (1): 137 - 140. 2004].     

琼胶酶研究进展

琼胶酶是一种多糖水解酶, 根据其降解琼脂糖的作用方式不同, 可以分为a- 琼胶酶(EC 3. 2.1. -) 和b- 琼胶酶(EC 3.2.1.81)。本文结合自己的研究, 从琼胶酶的生物学研究、酶的分类、晶体结构、催化机理以及酶的应用等几个方面综述了琼胶酶的研究进展。     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.