Synthesis and Properties of Trehalose-Based Flexible Polymers Prepared from Difurfurylidene Trehalose and Maleimide- Terminated Oligo(dimethylsiloxane) by Diels-Alder Reactions

Difurfurylidene trehalose (DFTreh) was synthesized from trehalose and furfural by an acetalization reaction. Maleimide-terminated dimethylsiloxane oligomers (DMS-BMI) were synthesized from amine-terminated dimethylsiloxane oligomers by condensation with maleic anhydride. Three types of DMS-BMI with different length were prepared. Trehalose-based polymers were synthesized by Diels-Alder reaction of DFTreh and DMS-BMI. The reaction proceeded at 40~70 °C to produce a polymer with a maximum weight average molecular weight of ~19,000. The thermal degradation temperature increased with the increase of the length of the oligo(dimethylsiloxane) units. The differential scanning calorimetry (DSC) measurements revealed the glass transition temperature (T_g) of the polymer at -130~-120 °C, and no distinct T_g not observed above room temperature in the DSC measurement. The polymer products are not liquid at room temperature, and solid films can be obtained by casting from solution, implying a phase-separated structure made up of soft and hard segments. The phase-separated structure was confirmed by transmission electron microscope (TEM) study. The DSC curve of the polymer showed a broad endothermic peak at 110~160 °C, suggesting that a retro-Diels-Alder reaction occurred. When a N,N-dimethylformamide solution of the polymer was kept at 100 °C and the resulting solution was analyzed by gel permeation chromatography (GPC), the molecular weight decreased and monomers appeared.     

Carbohydrate-active enzymes revealed in Coptotermes formosanus (Isoptera: Rhinotermitidae) transcriptome

Coptotermes formosanus is one of the most destructive wood-feeding termites. To understand the molecular mechanisms that regulate the development of the termite, a normalized C. formosanus cDNA library was constructed using mixed RNA isolated from workers, soldiers, nymphs and alates of both sexes. The sequencing of this library generated 131 636 expressed sequence tags (ESTs) and 25 939 assembled unigenes. The carbohydrate-active enzymes (CAZymes) revealed in this library were analysed in the present report. A total of 509 putative CAZymes were identified. Diverse cellulolytic enzymes were uncovered from both the host termite and from symbionts harboured by the termite, which were possibly the result of the high efficiency of cellulose utilization. CAZymes associated with trehalose biosynthetic and metabolic pathways were also identified, which are potential regulators of the physiological activities of trehalose, an important insect blood sugar. Representative CAZyme coding genes in glycoside hydrolase family 1 (GH1) were quantitatively analysed. The results showed that the five GH1 β-glucosidase genes were expressed differentially among different castes and one of them was female alate-specific. Overall, the normalized EST library provides a comprehensive genetic resource of C. formosanus and will serve a diverse range of research areas. The CAZymes represent one of the repositories of enzymes useful for physiological studies and applications in sugar-based biofuel production.     

海藻糖深低温保存外周血造血干细胞的可行性

背景：二甲基亚砜是目前造血干细胞深低温保存的经典保护剂，但其对细胞和患者均有一定的毒副作用。海藻糖是一种稳定的无毒副作用的非还原性双糖，已被广泛应用于红细胞、血小板和胚胎等的冷冻保存中。 目的：探讨海藻糖作为低温保存造血干细胞保护剂的可行性。 方法：外周血造血干细胞经重组人集落刺激因子动员后，用血细胞分离机采集连续单个核细胞，分为0.5 mol/L海藻糖组、1.0 mol/L海藻糖组、对照组。采用程序降温法液氮保存，冻存7 d后取出，立即置于40 ℃水浴箱内复苏。锥虫蓝拒染法检测细胞存活率；采用甲基纤维素半固体培养体系进行集落培养，计数粒-巨噬细胞集落形成单位的回收率；采用CD34-PE/CD45-FITC双标法，流式细胞仪检测CD34+细胞回收率。 结果与结论：与对照组比较，0.5，1.0 mol/L海藻糖组细胞存活率、粒-巨噬细胞集落形成单位回收率、CD34+细胞回收率均明显升高(P < 0.001)，且0.5 mol/L海藻糖组升高幅度尤为显著(P < 0.001或P < 0.01)。证实海藻糖对于短期内低温冻存的外周血造血干细胞有一定保护作用，浓度为0.5 mol/L的海藻糖保护冻存的造血干细胞效果较佳。 关键词：低温保存；粒-巨噬细胞集落形成单位；海藻糖；浓度；外周血造血干细胞；生物材料 doi:10.3969/j.issn.1673-8225.2010.12.008     

Feeding‐based RNA interference of a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is the most devastating rice insect pest to have given rise to an outbreak in recent years. RNA interference (RNAi) is a technological breakthrough that has been developed as a powerful tool for studying gene function and for the highly targeted control of insect pests. Here, we examined the effects of using a feeding‐based RNAi technique to target the gene trehalose phosphate synthase (TPS) in N. lugens. The full‐length cDNA of N. lugens TPS (NlTPS) is 3235 bp and has an open reading frame of 2424 bp, encoding a protein of 807 amino acids. NlTPS was expressed in the fat body, midgut and ovary. Quantitative real‐time PCR (qRT‐PCR) analysis revealed that NlTPS mRNA is expressed continuously with little change during the life of the insect. Efficient silencing of the TPS gene through double‐stranded RNA (dsRNA) feeding led to rapid and significant reduction levels of TPS mRNA and enzymatic activity. Additionally, the development of N. lugens larvae that had been fed with the dsRNA was disturbed, resulting in lethality, and the cumulative survival rates dropped to 75.56, 64.44, 55.56 and 40.00% after continuous ingestion of 0.5 µg/µl dsRNA for 2, 4, 7 and 10 days, respectively. These values were significantly lower than those of the insects in the control group, suggesting that NlTPS dsRNA may be useful as a means of insect pest control.     

膜分离技术提取海藻糖的工艺

采用超滤、纳滤操作对酵母抽提物进行处理，结果表明：操作压力、操作时间及料液体积流量对超滤有很大影响，所选MWCO为5000的膜件可去除96％以上的大分子蛋白质，起到纳滤预处理作用．采用MWCO为300的纳滤膜对超滤液进行浓缩纯化，海藻糖总提取率高达85．6％，大大高于传统方法．操作条件如进料压力、浓缩倍数及操作方式对纳滤过程均有很大影响。     

海藻糖对帕金森病氧化损伤保护作用及机制研究

目的研究海藻糖对1-甲基-4-苯基吡啶离子(MPP+)诱导的shsy5y细胞损伤的保护作用及机制。方法利用MPP+诱导损伤shsy5y建立帕金森病模型,分为对照组、损伤组、实验1组(海藻糖10mmol/L)、实验2组(海藻糖50mmol/L)和实验3组(海藻糖100mmol/L)。CCK-8法检测细胞存活率,利用二乙酰二氯氢化荧光素探针检测shsy5y细胞活性氧水平,hoechst 33342染色观察海藻糖对MPP+诱导的细胞凋亡保护作用,Western blot检测Nrf2、血红素氧化酶1(heme oxygenase-1,HO-1)抗氧化损伤蛋白表达。结果与对照组比较,损伤组shsy5y细胞存活率明显降低(P<0.01),Nrf2和HO-1表达明显降低(0.676±0.020 vs 1.000±0.067,P<0.05;0.546±0.049vs 1.000±0.048,P<0.01)。损伤组MPP+可以显著诱导shsy5y细胞发生凋亡,核碎裂,不同浓度实验组可以逆转这种诱导凋亡。实验1组Nrf2表达较损伤组明显升高,实验2组和实验3组Nrf2和HO-1表达较损伤组明显升高,差异有统计学意义(P<0.01)。损伤组shsy5y细胞内绿色荧光较对照组明显升高,不同浓度实验组随海藻糖浓度升高,shsy5y细胞内绿色荧光明显减少。结论海藻糖可以逆转MPP+作用的shsy5y细胞损伤,这一作用路径可能是通过促进Nrf2/HO-1信号通路激活,减少活性氧损伤,从而起到保护作用。     

海藻糖联合DMSO对大鼠带瓣主动脉的冷冻保护作用的研究

目的:研究非渗透性低温保护剂海藻糖联合应用二甲基亚砜,用于同种瓣的深低温储存,并与常规应用的二甲基亚砜的冻存效果进行比较。方法:分别以10%二甲基亚砜 0.1 mol/L海藻糖(实验组)和10%二甲基亚砜(对照组)作为冷冻保护剂,经程控梯度降温,液氮冻存大鼠的同种瓣6个月后复温,通过电镜、光镜观察和葡萄糖代谢率测定,对同种瓣的代谢功能和结构变化进行比较。结果:实验组的葡萄糖代谢率(20.570±1.789)g/(L.24 h);明显高于对照组的葡萄糖代谢率(18.621±1.842)g/(L.24 h)(P<0.01),并且其结构的破坏也较对照组轻(P<0.05)。结论:10%二甲基亚砜 0.1 mol/L海藻糖作为同种瓣的冷冻保护剂,其保护效果明显好于10%二甲基亚砜。     

人红细胞冻干保护剂配方及浓度的优化

本研究探讨不同红细胞冻干保护剂配方及浓度的变化对红细胞冻干-复水后回收率的影响。应用一系列不同浓度的聚乙烯吡咯烷酮（PVP）、海藻糖及不同渗透性保护剂组成的红细胞冻干保护剂保护冻干红细胞并检测冻干红细胞复冰后红细胞及血红蛋白的回收率．结果显示：荷载海藻糖的红细胞在添加不同浓度保护剂保护下各组的红细胞损失率差异具有显著性（P〈0．05或P〈0．01），其中以有PVP360的保护液组中的细胞损失最大（0．24％），含有PVP40且胞外海藻糖浓度为150mmol／L时，红细胞的损失最小（0．02％）．海藻糖浓度为150mmol／L与海藻糖浓度为50mmol／L的保护剂组之间存在统计学差异（P〈0．01）。冻干红细胞在添加不同PVP40浓度的冻干保护剂作用再水化后红细胞和血红蛋白回收率也不相同。15％PVP40＋150mmol／L海藻糖＋2％BSA在红细胞冻干中的保护效果最好，红细胞和血红蛋白回收率分别为（61．29±4．93）％，（62．49±5．91）％，与其它各组间存在显著差异（P〈0．01）。含有甘油的冻干保护液对红细胞冻干过程中的保护效果最好，红细胞和血红蛋白回收率分别为（65．97±4．52）％和（67．24±5．94）％，与其它渗透性保护剂组相比存在显著性差异（P〈0．01）。结论：红细胞冻干保护剂为0．8mol／L甘油＋15％PVP40＋150mmol／L海藻糖＋2％BSA是最佳保护剂浓度配方。