Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116R^F10. HCT116R^F10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116R^F10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116R^F10 cells. Interestingly, HCT116R^F10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.     

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri,Chordata)

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher’s) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name “B. belcheri EAS/AOS” (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.     

Evaporative light scattering mass detection for high-performance liquid chromatographic analysis of sucrose polyester blends in cooking oils

A high-performance liquid chromatographic method is described to determine the sucrose polyester (SPE) content in seven blends of cooking oils. Four gel-permeation chromatography (GPC) columns were used in series with an evaporative light scattering mass detector to separate the SPE from the acylglycerols in the final chromatogram. The SPE fraction was collected off the GPC column and injected onto a reverse-phase C-18 column for quantitation with sucrose octaacetate as an internal standard and a gradient of nonaqueous solvents as mobile phase. The chromatograms were interference-free, with only two sharp peaks appearing. The standards were linear from 500 to 5000 μg/mL with a correlation coefficient of r=0.999. The mean percent recovery (n=9) and standard deviation were 102±6.7. The detector could detect amounts as low as 5 μg SPE.     

Comparison of the construction of a 3-D model for human thromboxane synthase using P450cam and BM-3 as templates: implications for the substrate binding pocket

A 3-D model of human thromboxane A₂ synthase (TXAS) was constructedusing a homology modeling approach based on information fromthe 2.0 crystal structure of the hemoprotein domains of cytochromeP450BM-3 and P450cam. P450BM-3 is a bacterial fatty acid monooxygenaseresembling eukaryotic microsomal cytochrome P450s in primarystructure and function. TXAS shares 26.4% residue identity and48.4% residue similarity with the P450BM-3 hemoprotein domain.The homology score between TXAS and P450BM-3 is much higherthan that between TXAS and P450cam. Alignment between TXAS andthe P450BM-3 hemoprotein domain or P450cam was determined throughsequence searches. The P450BM-3 or P450cam main-chain coordinateswere spplied to the TXAS main chain in those sements where thetwo sequences were well aligned. These segments were linkedto one another using a fragment search method, and the sidechains were added to produce a 3-D model for TXAS. A TXAS substrate,prostaglandin H₂ (PGH₂) was docked into the TXAS cavity correspondingto the arachidonic acid binding pocket in P450BM-3 or camphorbinding site in P450cam. Regions of the heme and putative PGH2binding cavities in the TXAS model were identified and analyzed.The segments and residues involved in the active-site pocketof the TXAS model provide reasonable candidates for TXAS proteinengineering and inhibitor design. Comparison of the TXAS modelbased on P450BM-3 with another TXAS model based on the P450BM-3with another TXAS model based on the P450cam structure indicatedthat P450BM-3 is a more suitable template for homology modelingof TXAS.     

相转移催化合成蔗糖硬脂酸酯

采用相转移催化的基本原理和方法合成蔗糖硬脂酸酯,考察了反应时间、反应温度、原料的不同配比和催化剂用量对蔗糖硬脂酸酯产率的影响,得出反应时间为3 h,反应温度为95-100℃、硬脂酸甲酯与蔗糖的摩尔比为1:2.5,硬脂酸甲酯与PTC之比为1:0.07时为最佳配比。     

蔗糖酯对精炼大豆油氧化稳定性的影响

以不含抗氧化荆的精炼大豆油为原料,研究了加入蔗糖酯对其氧化稳定性的影响,结果表明：在低温时（烘箱法）,HLB13的蔗糖酯具有一定的延缓大豆油氧化的作用,其中0．5‰添加量（质量分数）时延缓氧化效果最好;高温条件下（110℃）HLB13、HLB11、HLB9的蔗糖酯在不同浓度条件下有一定的加速氧化作用．     

Surface disposition and stability of pest-interactive,trichome-exuded diterpenes and sucrose esters of tobacco

The precise physical location of trichome-exudate biochemicals on the plant surface is undoubtedly important in plant-pest interactions, perhaps particularly those involving fungal and bacterial pathogens that invade the plant through the epidermal layer. The chemical stability of exuded compounds is also important in this regard. Here we have studied these two aspects of trichome biology using the highly exuded tobacco line,Nicotiana tabacum, T.I. 1068. Particularly under high relative humidity growth conditions, sucrose esters (SE) were found to migrate from the exudate droplet around the gland down the trichome stalk to the epidermal cells below. Six days after labeling leaf midveins on plants grown in a high humidity environment, 29 and 71% of label found in SE were recovered with trichome glands and below gland regions, respectively. Corresponding disposition in the moderate humidity environment was 40 and 60%, respectively. Migration of less polar duvatrienediols (DVT) was less marked. Staining of SE with rhodamine B showed the occurrence of more extensive and physically different migration in the high humidity versus moderate humidity case. Both SE and DVT were stable between six and 18 days postlabeling, the period encompassing the time of maximum exudate formation through the beginning of tissue senescence. Our results suggest that even under conditions that avoid mechanical disturbance of tissue, SE and DVT are chemically stable, at least until senescence, and appear to migrate from the gland region to the epidermal surface, apparently according to their relative polarity.     

新型蔗糖基聚羧酸高效减水剂的合成与性能

以自制甲基丙烯酸蔗糖酯（MASE）、丙烯酸（AA）、甲基丙烯酸（MAA）、烯丙基磺酸钠和烯丙基聚乙二醇（A PEG ）为原料,以过硫酸钾为引发剂,自由基共聚法合成蔗糖基聚羧酸高效减水剂。研究了反应时间、蔗糖酯含量及引发剂用量对蔗糖基聚羧酸高效减水剂性能的影响。并通过流动度测试、红外光谱表征及黏度对减水剂的结构与性能进行了分析与比较。各组分物质的量nAA ：nMAA ：nSAS ：nAPEG ：nMASE ＝3∶1∶2∶1∶0．2,MASE含量为4．9wt．％,引发剂为单体用量1．9wt．％,反应时间为5h时合成的减水剂性能最好。在水灰比为0．29,折固掺量为0．3w t ．％,水泥净浆流动度达到340mm。     

基于HSI颜色空间的蔗糖结晶图像分割方法

针对煮糖过程蔗糖结晶图像的特点,采用颜色空间转换的方法,将图像从RGB颜色空间转换到HSI颜色空间,利用HSI颜色空间各分量相对独立性以及结晶颗粒和糖浆溶液的色调差异通过改进的大津法对H分量进行阈值分割,再通过数学形态学,中值滤波,孔洞填充,去除噪声颗粒,去除图像边界的非完整颗粒等方法进行后续处理,最终将结晶颗粒从复杂的图像信息中分割出来。实验证明该方法分割效果好,切实可行。