A colorimetric assay for screening microcystin class compounds in aquatic systems

Secondary metabolites produced by water-blooming cyanobacteria in eutrophic waters include some potent hepatotoxins, These compounds also have tumour-promoting properties, attributable to their inhibition and activation of protein phosphatases and kinases respectively. The inhibitory effect of these toxins on protein phosphatases have been employed in a commonly used radiometric assay, involving the use of a P-32-labeled substrate, for the detection and quantitation of these compounds. This paper investigates and describes a colorimetric method in which the activity of protein phosphatase 2A is determined by measuring the rate of colour production from the release of yellow p-nitrophenol using p-nitrophenyl phosphate as the substrate. Results of this study suggest that the colorimetric protein phosphatase inhibition assay is a simple, inexpensive tool for screening substances that may have tumour-promoting characteristics in aquatic systems. The detection limit of the colorimetric method is comparable to the radiometric assay. (C) 1998 Elsevier Science Ltd. All rights reserved.

Frankia的REP-PCR、RAPD-PCR鉴定及遗传多样性研究

本文采用溶菌酶法、CTAB法、微波法及CTAB+微波法等4种方法进行Frankia菌Cp11基因组DNA的提取。结果表明四种方式均可行，但以CTAB法及微波法最为有效。提取方法与REP-PCR带型间关系的研究表明，方法上的差异不会影响到REP-PCR带型的变化。对14株Frankia菌株作REP-PCR分析，并对之进行比较分类，证明REP-PCR方法不但能实现菌株间的差异鉴别，还能有效地进行Frankia菌的分类。这一结果与DNA同源相关性所得的分类结果具有良好的相关性。选用20个随机引物，对分自2个分类接种群的8株Frankia纯培养的总DNA进行随机扩增。其中引物OPW15和OPW16能扩增得到较为稳定的RAPD图谱。扩增产物分子量大都分布在0.5-4Kb之间。从稳定的RAPD扩增图谱看，Frankia菌间存在有丰富的遗传多样性；在选定适当引物情况下，能依据共同带型将Frankia菌化归为同一分类接种群。

rep-PCR对赤杨根瘤内局Frankia菌基因多样性研究

本文应用重复基因外回纹序列rep-PCR方法直接扩增长日山三种不同赤杨和云南不同地区早冬瓜根瘤内的Frankia菌基因，所获得的REP指纹图谱揭示Frankia菌具有丰富的基因多样性。研究结果表明Frankia菌多样性随地域，海拔，坡问不同而变：多样性指数与自然选抒）玉力大小成正相关：自然选择压力是产生和保持Frankia菌基因多样性的重要因子。同时发现Frankia菌基因型与地域有紧密关系：所观察的云南各地区中，感染高黎贡山旱冬瓜的Frankia菌基因多样性最为丰富。推测与高黎贡山旱冬瓜共生的Frankia可能作为种储备库，为其它地区的寄主植物提供了祖先菌株。在对长白山三种赤杨（东北赤杨，西伯利亚赤杨和色赤杨）根瘤内几百游殆菌基因多样性考察过程中发现，Frankia菌基因类型与寄主赤杨种型关系密切。同时海拔对感染东北赤杨的Frankia菌基因多样性也有一定影响。感染西伯利亚赤杨和色赤杨的Frankia菌享有相同的基因型，可能有较近的亲缘关系。

瓦山安息香中苯并呋喃促雌激素合成的机理研究

雌激素是人体内重要的激素之一，具有广泛的生理功能。雌激素缺乏与许多疾病相关，如卵巢功能低下，更年期综合征以及骨质疏松等；雌激素过剩也将导致某些疾病，如乳腺癌、卵巢癌、子宫内膜癌等。目前，如何降低肿瘤组织中的雌激素水平而达到治疗肿瘤的目的，已经得到广泛的研究，但促雌激素生成或调节卵巢功能药物或其相关研究则很少。 本实验室前期的研究发现，瓦山安息香属植物果实中的乙醇提取物具有促雌激素生成作用，通过活性追踪和结构鉴定，确认促E2 生成的主要成分为苯并呋喃类化合物。苯并呋喃类化合物的作用与芳香酶有关，但其确切的作用机理有待证实和深入研究。 为了探讨安息香苯并呋喃类化合物的促雌激素合成的作用机理，拟采用如下的实验方案： 1、细胞学方面，对小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、IGROV1 等细胞株，采用RT-PCR 和ELISA 方法研究芳香酶Aro基因的表达和雌二醇E2 的生成，芳香酶抑制剂Formestane 作为阳性对照，研究时效曲线和量效曲线，确定安息香苯并呋喃类化合物SP25 的有效浓度和作用时间。 2、RNAi 方面，设计合成了针对人芳香酶Aro基因的3 对RNAi 序列，转染入细胞，芳香酶促进剂Forskolin 和地塞米松、芳香酶抑制剂Formestane 作为阳性对照，采用实时定量PCR 技术，研究RNA 干扰后，安息香苯并呋喃类化合物SP25 对人芳香酶Aro基因表达水平瓦山安息香苯并呋喃促雌激素合成的机理研究的影响。 3、雌激素受体方面，设计一段ERE 的雌激素调控元件，构建重组荧光素酶报告基因载体，瞬时转染人乳腺癌细胞株MDA-MB-231，建立针对雌激素受体的报告基因筛选模型，观察安息香苯并呋喃类化合物SP25 对雌激素受体的选择性和亲和力，从受体水平考察安息香苯并呋喃类化合物SP25 促进雌激素生成的药理学机理。 实验结果显示： 1、分化后的小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7 、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-8 等细胞株具有芳香酶基因的表达。睾酮向雌二醇的转化能够被芳香酶抑制剂Formestane 所阻断，其中OVCAR-3 最适合进行下一步的RNAi研究。 2、RNAi 实验结果显示，设计的3 对RNAi 序列中R2 的干扰效果最强，相应的阴性对照C2 与R2 的表达量相差118 倍（24 小时）和19 倍（48 小时），显示R2/C2 这组序列可用于进一步的RNAi 试验。以R2 干扰OVCAR-3 细胞株，药物作用24、48 小时后，芳香酶抑制剂Formestane 与R2 相对表达量相比分别为0.83 倍和0.04 倍；芳香酶促进剂Forskolin 与R2 相对表达量相比分别为3.61 和1.84 倍；芳香酶促进剂地塞米松与R2 相对表达量相比分别为5.76 倍和3.49倍；苯并呋喃类化合物SP25 与R2 相对表达量相比分别为8.13 倍和4.59 倍。实验证实安息香苯并呋喃类化合物SP25 能够促进因RNAi 而发生基因沉默的人芳香酶Aro表达水平的上调。 3、雌激素受体实验结果显示，构建成功重组pERE-pGL3-promoter 荧光素酶报告基因载体和基于报告基因系统的雌激素受体激动剂或拮抗剂的细胞筛选模型。实验结果表明安息香苯并呋喃类化合物SP25 与雌激素受体ERα和ERβ亲和力选择性之比约为3：1 ，SP25通过与雌激素受体ERα结合作用其受体，刺激芳香酶的表达。 本课题通过RNA 干扰、ELISA、荧光实时定量PCR、报告基因筛选模型等技术手段，从细胞水平、蛋白酶水平和基因表达水平、雌激素受体水平等方面系统地研究了从瓦山安息香属植物果实中提取的苯并呋喃SP25 促进促雌激素生成的机理研究。试验结果显示苯并呋喃类化合物SP25 促雌激素生成的主要作用机制是直接促进芳香酶基因表达水平，以及与雌激素受体a 结合，刺激芳香酶活性。 Estrogen is an important hormone that has versatile physiologicalfunctions. Lack of estrogen will lead to many diseases such as lower ovarianfunction, climacteric syndrome and osteoporosis. Excessive estrogen alsoinduces breast carcinoma, oophoroma and endometrial carcinoma and otherdiseases. To depress the estrogen level in tumor tissue to cure carcinomawas widely studied, but there is only few studies reported on the induction ofestrogen and on the regulation of ovary function. We found that the extracts from seeds of Styrax perkinsiae couldpromote the synthesis of estrogen. The active compounds benzofurans wereidentified. Effect of benzofurans may be related to aromatase, but the mechanism was not clear. To reveal the mechanism of these benzofurans to promote estrogensynthesis, the following protocols were adopted: 1 Cytology: 3T3-L1 preadipocytes,human ovary carcinoma celllines OVCAR-3,OVCAR-4,OVCAR-5,OVCAR-8,IGROV1 andbreast carcinoma cell lines MCF-7 and MDA-MB-231 were usedto determine Aro gene expression and estrogen production withRT-PCR AND ELISA methods. Formestane, an aromataseinhibitor, was used as positive control. And dose-curve,time-curve and the effective concentration of SP25 were also studied. 2 Designed 3 pairs of RNAi for human aromatase gene, andtransfected into cell. Aromatase inducer Forskolin andDexamethasone, and aromatase inhibitor Formestane were usedas positive controls. We studied the change of Aro expressionlevel with SP25 by using real-time PCR after RNA interfering. 3 Estrogen Receptor: We constructed the recombined Luciferasereport vector and establish a screening system for estrogenagonist and antagon. With this system, we studied the affinity ofSP25 and estrogen receptor. Results: 1 Differentiated 3T3-L1 preadipocytes¡¢human ovary carcinomacell lines:OVCAR-3, OVCAR-4, OVCAR-8 and breast carcinomacell lines MCF-7, MDA-MB-231 had detected aromatase geneexpression.And OVCAR-3 is more suitable for further aromatasegene function research. 2 In RNAi assay, R2 has a strong interfering effcet in OVCAR-3 cellline, and ratio of C2 (the negative control) to R2 were 118 times(24 hours) and 19 times (48 hours). This means sucessful inRNA interfering. After R2 acted on OVCAR-3 cell line, the ratiosof formestane to R2 were 0.83 and 0.04 times, 5.76 and 3.49times (Dex), 3.61 and 1.84 times (forskolin) and 8.13 and 4.59times (sp25) after drug treated 24 or 48 hours respectively.These results indicated that SP25 can directly induce aromatasegene up-regulation. 3 We had constructed pERE-pGL3-promoter recombined vectorand the Luciferase report gene screening system. Luciferasereport gene assay showed that sp25 had a higher affinity with strogen receptor alpha than estrogen receptor beta, this indicated that SP25 can act on estrogen receptor and induce aromatase. Our results revealed that the mechanisms of benzofuran to promoteestrogen were the upregulation aromatase gene expression and promotion ofaromatase activity and have partially elective affinity with estrogen receptoralpha.

Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification

A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). The amplification could be finished in 60 min under isothermal condition at 64 degrees C by employing a set of four primers targeting the cap gene of PCV2. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of five copies per tube of purified PCV2 genomic DNA. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1 (PCV1), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection rate of PCV2 LAMP for 86 clinical samples was 96.5% and appeared greater than that of the PCR method. The LAMP assay reported can provide a rapid yet simple test of PCV2 suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction. (c) 2008 Elsevier B.V. All rights reserved.

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of '' Tail DNA (%)'' (TD) and "Olive tail moment" (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radio sensitivity