一种新的以影像为基础的细胞增殖和细胞毒性检测方法

3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑盐)（MTT）比色法是传统上检测细胞增殖和细胞毒性的常用方法.
CloneSelectTM成像系统是一种以影像为基础的用于分析细胞生长的可视检测系统.本研究采用人结直肠癌HCT116细胞系,运用CloneSelect成像系统和MTT方法分别检测药物阿的平的细胞毒性,并采用Bland Altman作图法比较两种实验方法获得的pEC50值,分析两种研究方法获得的结果的一致性. 结果表明,CloneSelectTM成像系统和MTT法获得的pEC50值具有较好的一致性.与MTT方法相比,基于影像的CloneSelectTM成像分析技术检测快速、无损伤且结果更准确,获取资料不损伤细胞,允许后续其它时间点或动力学检测. 研究提示,这种新的以影像为基础的检测技术可以替代MTT方法,用于分析不同药物的抗细胞增殖活性.     

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。     

豌豆蚜触角转录组化学感受蛋白的鉴定、表达和结合特性分析

[目的]本研究旨在鉴定豌豆蚜Acyrthosiphon pisum触角转录组中化学感受蛋白(chemosensory protein,CSP)基因,明确触角中高表达的豌豆蚜CSP蛋白与蚜虫报警信息素、性信息素以及植物挥发物的分子结合特性.[方法]通过对豌豆蚜成蚜触角进行转录组测序,鉴定触角中候选CSP基因;采用RPKM...     

A rapid method for determining dynamic binding capacity of resins for the purification of proteins

Biolayer interferometry is a novel method for quantifying macromolecules, such as proteins, in solution. The presence of other, non-binding molecules does not interfere with quantification, which allows one to measure the concentration of the molecule of interest in a crude mixture. Here we apply this method to determining the dynamic binding capacity of affinity resins.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

A mutation at the Ala122 position of acetohydroxyacid synthase (AHAS) located on chromosome 6D of wheat: improved resistance to imidazolinone and a faster assay for marker assisted selection

A new mutation at the acetohydroxyacid synthase (AHAS) locus on chromosome 6D of wheat was analyzed in detail because it conferred an improved resistance to the imidazolinone group of herbicides. Sequence analysis showed that the mutation was at the Ala122 position (A122T), a position in AHAS which has not to date been identified in imidazolinone resistant wheat lines even though the position has been identified in other plants and is associated with resistance. An allele-specific assay for the mutation (in the wheat line Brookton-8) was developed and used in a genetic analysis. Two mapping populations were analysed and the doubled haploid progeny from the cross Brookton-8 × Clearfield STL proved to be most informative. The AHAS^Ala122 mutation (A122T) was allelic to the AHAS^Ser653 mutation (S653N) in Clearfield STL (Imi1, on chromosome 6D) and hence was assigned to the chromosome 6D locus. The analysis of the doubled haploid lines in the mapping population demonstrated the greater resistance conferred by the A122T mutation because lines from the same cross and carrying either the A122T or S653N mutations could be directly compared. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): Effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T₁₄ and T₂₈, while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T₂₈ only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk mouse lymphoma cells

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects. Short-term tests for genotoxicity of furan in mammalian cells are inconclusive, little is known for BDA. We investigated BDA generated by hydrolysis of 2,5-diacetoxy-2,5-dihydrofuran for genotoxicity in L5178Y tk^+/− mouse lymphoma cells using standard procedures for the comet assay, the micronucleus test, and the mouse lymphoma thymidine kinase gene mutation assay, using 4-h incubation periods. Cytotoxicity was remarkable: cell viability at concentrations ≥50 μM was reduced to <50%. In the dose range up to 25 μM, viability was >90%. Measures of comet-tail length and thymidine–kinase mutant frequency were increased 1.6- and 2.4-fold above control, respectively. Analysis of three fully independent replicates with a linear mixed-effects model showed a highly significant increase with concentration for both endpoints. Compared to methyl methanesulfonate used as a positive control, BDA was of similar potency with respect to genotoxicity, but it was much more cytotoxic. Furan added to cell cultures at doses that resulted in time-averaged effective concentrations of up to 3100 μM was neither cytotoxic nor genotoxic. A potential cross-linking activity of BDA was investigated by checking whether gamma radiation-induced DNA migration in the comet assay could be reduced by pre-treatment with BDA. In contrast to the effect of the positive control glutaraldehyde, BDA treatment did not reduce the comet tail length. On the contrary, an increase was observed at ≥100 μM BDA, which was attributable to early apoptotic cells. Although BDA was found to be a relatively potent genotoxic agent in terms of the concentration necessary to double the background measures, cytotoxicity strongly limited the concentration range that produced interpretable results. This may explain some of the inconclusive results and indicates that non-genotoxic effects must be taken into account in the discussion of the modes of toxic and carcinogenic action of furan.