Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Genotoxicity assessment of Copaiba oil and its fractions in Swiss mice

Copaiba oil-resin, extracted from the trunk of Copaifera, and traditionally used in folk medicine in the treatment of various disorders, has been shown to be an effective antiinflamatory, antitumor, antitetanus, antiseptic and anti-blenorrhagea agent. As, there are few studies evaluating its genotoxicity, this aspect of the commercial oil-resin, and its volatile and resinous fractions, were evaluated in mice by comet assay and micronucleus (MN) test. A single dose of oil resin, volatile or resin fractions (500; 1,000 or 2,000 mg/kg b.w.) was administered by gavage. The chemical compositions of Copaiba oil resin and its fractions was analyzed by gas chromatography. According to comet assaying, treatment with either one did not increase DNA damage, and as to MN testing, there was no alteration in the incidence of micronucleated polychromatic erythrocytes. Chromatographic analysis of the oil-resin itself revealed sesquiterpenes, diterpenic carboxylic acid methyl esters and high levels of β-caryophyllene. Thus, it can be assumed that the oil resin and volatile and resinous fractions from the commercial product are not genotoxic or mutagenic.     

昆虫神经毒性酯酶活性的测定

首次报道了针对昆虫建立的神经毒性酯酶(NTE)活性检测方法以及据此法所测得的棉铃虫幼虫的NTE活性。将测定脊椎动物NTE活性的方法改进并微量化以适用于无脊椎动物昆虫体内NTE活性测定。对于棉铃虫幼虫,该法测得其头部、中肠和脂肪体等3个部位的NTE活性分别为5.30,1.40和14.50nmolminmgprotein。     

玉米转录因子zmCBF1的凝胶阻滞分析

凝胶阻滞试验是分析核酸与蛋白质相互作用的有效方法,在转录因子的功能分析中得到了广泛应用。以玉米转录因子zmCBF1与顺式元件CRT的结合为例,建立了用于转录因子分析的GRA/EMSA实验体系,并对其在应用中可能出现的问题进行了分析。     

Reaction of Trigonopsis variabilis d-amino acid oxidase with 2,6-dichloroindophenol: kinetic characterisation and development of an oxygen-independent assay of the enzyme activity

2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d-amino acid oxidase (TvDAO) with d-methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen (K_m ≈ 0.7 mmol L⁻¹).     

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67 ± 19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations ≥100 μM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations ≥300 μM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by DL-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (≥1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.     

从噬菌体展示抗体文库筛选对虾白斑综合征病毒的单链抗体

对虾白斑综合征是一种严重危害对虾养殖业的病毒性疾病.由于目前对其病原体对虾白斑综合征病毒（WSSV）的研究不够深入,所以对WSSV的有效防治仍然是一大难题.为此,用完整的对虾白斑综合征病毒粒子作为靶抗原固相包被,淘选噬菌体展示单链抗体文库,得到两个能够与WSSV结合的单链抗体：E2和H4.单链抗体H4能够结合病毒并抑制病毒对原代培养的对虾淋巴细胞的感染,这些结果表明此单链抗体具有开发为诊断试剂盒和抗病毒药物的潜力.     

棉花脱水应答元件(DRE)结合蛋白GhDBP1的原核表达、纯化及其DNA结合活性

棉花是一种重要的经济作物,其生产和产量要受到干旱、低温和高盐等环境胁迫的影响,因此提高棉花对这些胁迫的抗性非常重要.脱水应答元件(DRE-dehydrationresponsiveelement)结合蛋白(DBP)在调节植物对环境胁迫的抗性中起到非常重要的作用.而且过量表达DBP类基因的转基因植株能够很好抵抗这些环境胁迫,所以研究棉花中此类DRE元件结合蛋白对棉花生产有非常重要的意义.在以前的工作中,从棉花中分离一个DBP基因,命名为GhDBP1并在转录水平上分析它在棉花植株中的表达特征.在研究中,报道了GhDBP1的原核表达、纯化和它的DNA结合特性.GhDBP1基因的编码区用PCR技术扩增出来插入到原核表达载体pET28a中,并转化到大肠杆菌菌株BL21(DE3)中.经过IPTG诱导,GhDBP1融合蛋白在BL21(DE3)菌株中成功进行表达.利用Ni-NTA亲和层析技术得到了纯化的融合蛋白.在非同位素的凝胶滞留实验中,纯化的GhDBP1融合蛋白能够结合到含有DRE元件的DNA片段上.另外,用SWISS-MODEL软件对GhDBP1蛋白的DNA结合区的三维结构进行了计算机模拟.模拟的结果显示,GhDBP1蛋白的DNA结合区的主链结构和折叠模式与已知的拟南芥GCC盒结合蛋白AtERF1的DNA结合区结构很相似.这些结果显示了GhDBP1是一个脱水应答元件(DRE)结合的转录因子,并可能运用与AtERF1的DNA结合区相似的结构和它的目标序列脱水应答元件(DRE)相结合.     

两个水稻GDP-甘露糖-3’，5＇-异构酶基因特征及表达模式的研究

GDP-甘露糖-3’,5’-异构酶（GME）可以催化GDP-甘露糖转化为左旋GDP-半乳糖,该反应对于高等植物体内抗坏血酸的合成是非常重要的．但目前在分子水平上还没有对GME基因进行研究的报道．通过逆转录PCR（RT-RCR）技术从水稻成熟叶片中克隆到两个GME基因的cDNA序列,并与其他植物物种中的GMEs进行比对,结果显示,GME基因在所有植物物种中高度保守,尽管进化树分析表明单子叶植物GMEs和双子叶植物GMEs在进化上相互独立．同时,分析这两个水稻GME基因的剪切模式揭示了二者也存在高度相似性．采用半定量RT-PCR技术对两个GME基因在不同组织和不同胁迫条件下的表达模式进行研究表明,OsGME1基因在冷胁迫条件下表达水平上调,这和先前水稻冷胁迫蛋白质组学研究的结果是一致的．而OsGME2和OsGME1基因在用赤霉素处理条件下表达水平均下调,暗示赤霉素可能通过调节GME基因的表达来调控植物体内的抗坏血酸合成．