耻垢分枝杆菌海藻糖合成酶基因TreS的克隆及其在大肠杆菌中的表达

耻垢分枝杆菌(Mycobacterium smegmatis)在环境胁迫下具有合成海藻糖的能力[1-2].实验采用PCR的方法,克隆了来源于耻垢分枝杆菌的一段核苷酸序列,与已报道的海藻糖合成酶有60%以上同源性,推测该基因的编码产物具有海藻糖合成酶的活性,为进行其功能验证而在大肠杆菌中进行了表达.目的蛋白以可溶性蛋白为主,约占细胞可溶性总蛋白48%.为目前相关报道的最高值.经活性测定,表达的重组酶无需复性,能够催化麦芽糖生成海藻糖,在20℃反应20 h对麦芽糖的转化率可达61%,具有较高的应用价值.     

一株产海藻糖合成酶嗜中温芽孢杆菌的鉴定及其酶学性质

由长白山温泉附近土壤中筛选得到产海藻糖舍酶菌株，为革兰氏阳性菌，具有芽孢和鞭毛，其最适生长温度为55℃，对这一菌株进行生理生化指标测定。进一步对该菌株所产海藻糖合酶的酶学性质进行研究，酶反应最适作用pH值为7．0，在pH6．6～7．4时该酶稳定，最适作用温度为35℃，在25-45℃时该酶可稳定保存。     

从新鲜面包酵母中提取海藻糖

研究了加热预处理对从新鲜酵母中提取海藻糖的影响,指出较高温度预处理能提高海藻糖提取率,加热预处理条件１０５℃,６０ｈ,能使海藻糖提取率比从新鲜酵母提取增加１．２倍．而对于已加热预处理的酵母,可以用水瞬时提取,不受时间和温度的限制     

耐高渗酿酒酵母的代谢流分析

研究了指数生长期的酿酒酵母 (Saccharomyces cerevisiae) 在高渗胁迫下的生理代谢.甘油和海藻糖是酿酒酵母的主要相容性溶质.在高渗环境下,胞外酒精质量浓度、甘油质量浓度、海藻糖质量浓度分别较对照提高100%、400%和11%.代谢流分析表明,高渗环境下,从节点6-磷酸葡萄糖合成海藻糖的碳流较常规培养增加了47.3%;从节点磷酸二羟丙酮和3-磷酸甘油醛合成甘油的碳流增加了55.7%;从节点丙酮酸流向乙醇的碳流增加了300%,而流向三羧酸 (TCA) 循环的流量减少了21.7%.     

海藻糖对低温保存皮肤组织中β-肌动蛋白的作用研究

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对低温保存皮肤组织中β-肌动蛋白的影响.方法 将新鲜成人皮肤分为5组,分别将海藻糖/二甲基亚砜、二甲基亚砜/丙二醇、二甲基亚砜/无血清角质细胞培养基、DMEM作为冷冻保护剂,液氮冻存14d后复温,采用免疫组织化学染色、Western-blot、RT-PCR等方法,比较各组皮肤组织中β-肌动蛋白的蛋白和基因表达变化.结果 0.5M 海藻糖/二甲基亚砜作为保护剂所冷冻的皮肤,复温后其β-肌动蛋白的蛋白和基因表达明显高于其他组.结论 海藻糖与二甲基亚砜联合应用能较好的保护皮肤组织中的β-肌动蛋白,整形外科在常规应用保护剂时可以在渗透性保护剂中加入海藻糖.     

海藻糖对低温保存皮肤中E钙黏素表达的影响

目的 将非渗透性低温保护剂海藻糖用于人类皮肤的低温储存,观察海藻糖对冻存皮肤组织中E钙黏素的影响,探讨海藻糖的低温保护作用.方法 新鲜成人皮肤组织分为新鲜对照组、海藻糖-二甲基亚砜(T-D)组、二甲基亚砜-丙二醇(D-P)组均在-196 ℃液氮冻存7 d,第14天复温,免疫组织化学染色对各组间皮肤进行比较.结果 与新鲜皮片组相比,0.5 mol/L T-D能够很好保护皮肤组织,E钙黏素的蛋白表达量与新鲜皮肤组相似.结论 海藻糖与二甲基亚砜联合应用能较好地保护皮肤组织E钙黏素,其对皮肤组织的保存效果优于传统的低温保护剂.     

海藻糖用于小鼠卵母细胞玻璃化冷冻的研究

目的 探讨海藻糖对小鼠卵母细胞玻璃化冷冻的影响. 方法 采用12只小鼠控制性超排卵获得的360枚成熟的卵母细胞,分别放入含1.0、0.5、0.3及0 mol/L的海藻糖中孵育3h.部分卵母细胞进行碘化丙啶(PI)染色;320枚卵母细胞进行玻璃化冷冻和复苏,并进行体外受精,观察卵母细胞成活率、受精率、卵裂率及囊胚形成率. 结果 0.3 mol/L海藻糖组复苏率、受精率与对照组无显著差异(92.59％ vs.90.90％,70.67％ vs.61.43％),但是卵裂率、囊胚形成率均高于对照组(83.02％ vs.44.19％,72.72％ vs.47.37％)(P＜0.001);0.5 mol/L海藻糖组复苏率、受精率、囊胚形成率与对照组无显著差异,但是卵裂率高于对照组(65.22％ vs.44.19％)(P＜0.05);0.3 mol/L海藻糖组囊胚形成率高于0.5 mol/L海藻糖组(72.72％ vs.50.00％)(P＜0.001);1.0 mol/L海藻糖组卵母细胞复苏率、受精率、卵裂率、囊胚形成率均低于对照组低(P＜0.001). 结论 一定浓度范围的海藻糖对小鼠卵母细胞的玻璃化冷冻有保护作用,0.3 mol/L海藻糖浓度比较适合小鼠卵母细胞玻璃化冷冻.     

海藻糖对液氮深低温保存的同种瓣caspase-3表达的影响

目的 探讨海藻糖作为深低温冷冻保护剂对液氮保存的同种带瓣大动脉组织细胞caspase-3表达的影响.方法 在液氮中冻存时间点分为12、15、18个月,依据冻存液不同分为5组,分别是:0.1 mol/L DMSO(对照组),0.1 mol/L海藻糖(实验组1),0.1 mol/L海藻糖+0.1 mol/L DMSO (实...     

酵母抽提物超滤制备海藻糖工艺条件的优化

利用响应面分析法对影响酵母抽提液中总氮截留率和海藻糖透过率的主要因素进行分析,优化超滤工艺,得到最佳超滤条件:料液质量浓度10 g／dL,料液温度42℃,操作压力0.31MPa,在最佳超滤条件下得到酵母抽提物蛋白质截留率96.5%,海藻糖透过率94.8%,所得RSA图可直观地反映各因素与蛋白氮截留率和海藻糖透过率的关系.     

海藻糖在同种瓣低温保存中的保护作用

目的探讨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＋0．1mol／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，电镜、光镜观察、内皮细胞活力及葡萄糖代谢率测定，比较同种瓣的组织活性、代谢功能和结构变化。结果实验组的葡萄糖代谢率为（20．570±1．789）晷／L·d^-1明显高于对照组（18．621±1．842）g／L·d^-1（P〈0．01），内皮细胞活力（77．75±6．60）％也明显高于对照组（69．81±4．40）％（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＋0．1mol／L海藻糖作为同种瓣的冷冻保护剂，其保护效果明显优于10％二甲基亚砜。     

Evaluation of a new solution containg trehalose for twenty-hour canine lung preservation

We examined the efficacy of two new preservation solutions containing trehalose-an extracellular type (ET-K) of solution and an intracellular type (IT-K) of solution — in relation to that of Euro-Collins (EC) solution in 20-h canine lung preservation. Canine lungs were flushed with one of the three solutions (n=5 for each solution) after pretreatment with PGE₁ (20 g/kg) and were stored for 20 h at 4°C. The left lungs were transplanted and evaluated to 6 h post transplant. In the ET-K group, the arterial oxygen tension after reperfusion was significantly higher than in the IT-K and EC groups. The pulmonary vascular resistance, wet/dry weight ratio, and histological evaluation of each transplanted lung in the ET-K group were also better than in the IT-K and EC groups. This indicates that ET-K solution is useful for 20-h preservation of canine lung grafts.     

海藻糖对脂质膜保护作用的荧光分析

以蛋黄卵磷脂（EPC）为模型，通过反映磷脂头部基团运动情况的荧光各向异性的变化，研究海藻糖对蛋黄卵磷脂（EPC）脂质膜的保护作用，并对其机理进行探讨，试验表明：荧光各向异性A值曲线随温度变化呈抛物线形状。     

用薄层层析法分析海藻糖

从制备薄层的各种硅胶、调浆剂着手制备硅胶薄板，然后选择展开剂，解决海藻糖不易与其他双糖分开的难题，建立了一种理想的海藻糖薄层层析定性分析方法。用本方法可检出２μｇ海藻糖，简单，快速，灵敏。     

从活性干酵母中提取海藻糖的工艺

提出了一条活性干酵母中提取海藻糖的工艺路线。通过控制抽提温度、乙醇浓度以及抽提时间，得出较佳的提取工艺条件。用阴阳离子交换柱去除杂质，同时起到一定的脱色作用，进而运用超滤进行澄清。最后经浓缩、结晶以及干燥，制成海藻糖产品。其得率可达：每１００ｇ活性干酵母产１４．５ｇ海藻糖。     

海藻糖对面包酵母耐贮存力的影响

初步研究了面包酵母胞内海藻糖含量对其耐贮存力的影响，探讨了提高鲜酵母胞内海藻糖含量的培养措施。研究结果表明，海藻糖含量越高，面包酵母的耐贮存力越强。采用提高海藻糖含量的培养技术进行流加培养，发酵结束时面包酵母中海藻糖含量从９．７ｍｇ／ｇ提高到１９ｍｇ／ｇ．４℃贮存１８ｄ后，面包酵母的发酵活力由１１７０ｍｌ变为１０４０ｍｌ（仅下降了１１％），表现出较优的耐贮存力。     

Evaluation of synergic effects of iodixanol and trehalose on cryosurvival of electroejaculated ram semen

The primary aim of the study was to investigate whether iodixanol and trehalose would have a synergic effect on the cryosurvival of electroejaculated ram semen. Tris-based diluter was used to prepare 9 different extenders by the addition of iodixanol or trehalose alone or varying combinations of these substances. Diluters were prepared as follows: Tris (control), Io5 (5% iodixanol), Tr25 (25 mmol/L trehalose), Tr50 (50 mmol/L trehalose), Tr50 + Io1.25 (50 mmol/L trehalose and 1.25% iodixanol), Tr50 + Io2.5 (50 mmol/L trehalose and 2.5% iodixanol), Tr50 + Io5 (50 mmol/L trehalose and 5% iodixanol), Tr25 + Io5 (25 mmol/L trehalose and 5% iodixanol) and Tr12.5 + Io5 (12.5 mmol/L trehalose and 5% iodixanol). Supplementation of the freezing extender with trehalose or iodixanol alone supported the protection of both morphological and functional integrity of ram spermatozoa and total motility at 1 and 4 hr post-thawing respectively. However, beyond these positive effects, the combination of trehalose (25 mmol/L) and iodixanol (5%) significantly increased post-thaw sperm longevity and motion properties at the end of 4-hr incubation. The results of the study clearly showed that there was positive synergic effect of iodixanol and trehalose on cryosurvival of ram semen.     

Trehalose enhances osmotic tolerance and suppresses lysophosphatidylcholine‐induced acrosome reaction in ram spermatozoon

This study was aimed to investigate the influence of trehalose on osmotic tolerance and the ability of ram spermatozoon to undergo acrosome reaction induced by lysophosphatidylcholine (LPC). In experiment 1, the diluted ejaculates were exposed to anisosmotic fructose solutions (70, 500, 750 and 1000 mOsm l^?1) with or without 50 mm trehalose. The presence of trehalose in hyperosmotic conditions enhanced (P < 0.05) the percentage of live, live‐intact and intact spermatozoa. Similarly, trehalose enhanced (P < 0.05) the live and live‐intact spermatozoa during hypo‐osmotic conditions. In experiment 2, the centrifuged ejaculates were diluted with TCG only or TCG containing either 50 or 100 mm trehalose. The acrosome reaction was induced by LPC. The percentage of acrosome‐reacted spermatozoon was less (P < 0.05) in trehalose‐supplemented groups compared to control. In experiment 3, the ejaculates were cryopreserved in an extender containing 0 mm (control), 50 mm or 100 mm trehalose. Supplementation of extender with trehalose, either 50 mm or 100 mm , enhanced the cryosurvival rate (P < 0.05) compared to the control. In conclusion, the presence of trehalose in anisosmotic conditions enhances the osmotic tolerance, cryosurvival rate of ram spermatozoon and suppresses their ability to undergo LPC and cryo‐induced acrosome reaction.     

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。     

Trehalose sustains a higher post‐thaw sperm motility than sucrose in vitrified human sperm

One of the cryopreservation methods that best preserves sperm function is vitrification. However, comparative studies have not been performed to evaluate the effect of nonpermeable cryoprotectors on sperm function for prolonged periods of time post‐devitrification. These times are necessary, especially in in vitro fertilisation and intrauterine insemination, for gamete interaction and then fertilisation to occur, while maintaining motility to arrive at the fertilisation site. In this study, sucrose (.25 m ) and trehalose (.1 and .05 m ) were compared in essential parameters like motility and plasma membrane integrity for 12 hr. Post‐devitrification sperm motility using .1 m trehalose was 68.9%, higher than that obtained with .05 m trehalose (59.9%, p < .0081) and .25 m sucrose (57.9%, p < .0002). Similar results were obtained at 6 and 12 hr with .1 m trehalose (58.0% and 42.3% respectively) compared to .05 m trehalose (p < .0184 and p < .033) and .25 m sucrose (p < .0001 and p < .0012).There was no difference between .25 m sucrose and .05 m trehalose. Membrane integrity was best preserved at time 0 by .1 m trehalose (p < .05), but there was no significance at 6 and 12 hr compared to sucrose. Our results suggest that for assisted reproduction techniques that require motile spermatozoa for a longer period of time, use of .1 m trehalose is recommended in the sperm vitrification technique.