Changes in trehalase activity and trehalose level during Ascaris suum (Nematoda) embryogenesis

Summary The levels of trehalose and the activity of trehalase during the development of Ascaris suum eggs were investigated. The level of trehalose in the zygote was high (2.96 ± 0.07 mg/g). During cleavage of eggs, it decreased (0.91 ± 0.35 mg/g). A higher concentration of the sugar was recorded at the blastula and during gastrulation, but it did not reach the uncleaved eggs level. In the early motile larvae, the concentration of trehalose was high (4.58 ± 2.01 mg/g). It decreased with development of L₁ larvae (3.10 ± 1.47 mg/g). A rapid increase in trehalose reserves was observed between the L₁ and L₂ stages. The highest content of trehalose was found in invasive L₂ larvae (5.78 ± 1.39 mg/g). The activity of trehalase at the zygote stage was high (560.22 ± 322.31 U/mg). It decreased at the beginning of cleavage. It was the lowest at the 4–6-cell stage (189.76 ± 114.97 U/mg). An increase in the enzyme activity occurred after reaching the blastula stage (348.44 ± 343.34 U/mg). The highest trehalase activity was recorded during the L₁ larvae stage (635.72 ± 251.16 U/mg). The activity of that enzyme was about three times lower in the invasive stage larvae than in the L₁ larvae.     

Changes in external trehalase activity during human serum-induced dimorphic transition in Candida albicans

Yeast-like cells (blastoconidia) of Candida albicans growing exponentially on a glucose-containing medium (YPD) exhibited low external trehalase activity and stored a negligible amount of intracellular trehalose. The addition of human serum at 37 degrees C to exponential cultures promoted a high degree of germ-tube formation with no significant changes in trehalase activity or trehalose content. In contrast, stationary cells accumulated a large amount of trehalose, while external trehalase remained at a low and practically constant level. However, resting cultures were unable to enter the dimorphic program, except when they were supplemented with fresh YPD and serum together. Only under these conditions was trehalase activated and trehalose hydrolyzed. Specific inhibition of external trehalase by validoxylamine A caused a certain delay in, and a lower level of, germ-tube formation, but did not totally block the dimorphic conversion. These results suggest that external trehalase is not involved in the serum-induced morphological transition in C. albicans.     

Continuous Release of rh-Interferon α-2a from Triglyceride Implants: Storage Stability of the Dosage Forms

Tristearin implants containing polyethylene glycol 6000 (PEG) were shown to be a promising platform for the delivery of pharmaceutical proteins for periods up to 1 month. The objective of this study was to investigate the storage stability of the lipid devices, as long-term storage stability ensuring acceptable shelf-life can be considered the most important parameter for commercially viable sustained-release dosage forms. Rh-Interferon α-2a was stabilized by a lyophilization process using either trehalose or hydroxypropyl-β-cyclodextrin as stabilizer. Tristearin implants containing the lyophilized protein material and 10% PEG were stored over 3 months and 6 months, both at 4°C and room temperature, before release studies were initiated. Data from stored implants demonstrated trehalose not to be effective to provide full protein stabilization during long-term storage of the lipid matrices, this was apparent from both the reduced total drug level liberated and the release of aggregated specimen compared to the situation immediately after implant manufacture. In contrast, hydroxypropyl-β-cyclodextrin (HP-β-CD) exhibited a high potential for protein stabilization within the matrices during both storage and release. Generally, 95% of the incorporated protein was delivered continuously within 1 month in monomeric form, even after 6 months' storage of the implants at room temperature.     

海藻糖对液氮保存同种带瓣大动脉组织结构的影响

目的观察3种不同保护剂对液氮冻存同种带瓣大动脉组织结构影响,寻求最佳冷冻保护剂。方法单独使用0．1mol／L二甲亚砜（对照组）及单独使用0．1mol／L海藻糖（实验组1）和0．1mol／L二甲亚砜＋0．1mol／L海藻糖（实验组2）作为冷冻保护剂,用光镜及电镜对新鲜组织及液氮保存6、9、12个月的带瓣大动脉进行形态学观察。结果随着保存时间的延长,3组结构破坏均逐渐加重。光镜下观察,保存超过6个月后,3组形态学与新鲜组织比较均有改变（d=17～30,P〈0．01）,3组之间比较差异有显著意义（d=6～13,P〈0．01、0．05）。电镜下观察,保存6、9、12个月时,对照组的同种带瓣大动脉组织的超微结构改变十分明显,实验组1改变较对照组轻,实验组2的结构改变最轻。结论海藻糖对液氮保存的带瓣大动脉有较好的保护作用;单独使用0．1mol／L海藻糖保护效果优于单独使用0．1mol／L二甲亚砜,二者联合应用保护效果明显好于单独使用。     

异氟醚对Aβ25-35诱导大鼠PC12细胞氧化应激损伤的影响及海藻糖的保护作用

目的:探讨吸入麻醉药异氟醚对β淀粉样蛋白25-35(Aβ25-35)诱导大鼠PC12细胞氧化应激损伤的影响,阐明海藻糖对其可能的预防及保护作用。方法:将大鼠PC12细胞随机分为正常对照组(Control组)、异氟醚组(Iso组)、Aβ25-35组(Aβ组)、异氟醚+Aβ25-35组(Iso+Aβ组)、异氟醚+Aβ25-35+海藻糖组(Iso+Aβ+Tre组)和海藻糖组(Tre组)。正常对照组,PC12细胞给予正常细胞培养基培养;Iso组,PC12细胞给予2%异氟醚;Aβ组,PC12细胞给予10 μmol·L^-1 Aβ25-35;Iso+Aβ组,PC12细胞给予2%异氟醚和10 μmol·L^-1 Aβ 25-35;Iso+Aβ+Tre组,PC12细胞给予2%异氟醚、10 μmol·L^-1 Aβ25-35和200 mmol·L^-1海藻糖;Tre组,PC12细胞给予200 mmol·L^-1海藻糖。采用MTT法检测细胞存活率;Hoechst 33342荧光染色检测细胞凋亡率;二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光法检测细胞中活性氧(ROS)水平;化学发光法测定细胞中丙二醛(MDA)水平,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及过氧化氢酶(CAT)活性。结果:与正常对照组比较,Iso组、Aβ组和Iso+Aβ组PC12细胞凋亡率明显升高(P<0.05或P<0.01),细胞存活率明显降低(P<0.05或P<0.01),细胞中ROS和MDA水平(P<0.05或P<0.01)明显升高,细胞中SOD、GSH-Px和CAT活性明显降低(P<0.05或P<0.01);与Iso和Aβ组比较,Iso+Aβ组细胞凋亡率明显升高(P<0.05),但细胞存活率明显降低(P<0.05),细胞中ROS、MDA水平(P<0.05)明显升高,细胞中SOD和GSH-Px和CAT活性明显降低(P<0.05);与Iso+Aβ组比较,Iso+Aβ+Tre组细胞凋亡率(P<0.05)明显降低,细胞存活率明显升高(P<0.05),细胞中ROS和MDA水平明显降低(P<0.05),细胞中SOD、GSH-Px和CAT活性明显升高(P<0.05)。结论:吸入麻醉药异氟醚能够加剧Aβ25-35诱导PC12细胞氧化应激损伤和细胞凋亡,海藻糖能够通过抗氧化和抗凋亡作用拮抗异氟醚的细胞毒性。     

异氟醚对转APP基因小鼠脑内海马神经元蛋白质损伤和蛋白聚集的影响

目的：观察吸入麻醉药异氟醚对转APP基因小鼠脑内海马神经元蛋白质损伤和蛋白聚集的影响,并探讨海藻糖对异氟醚诱导的神经毒性的干预作用。方法：60只12月龄转APP基因小鼠随机分为对照组、异氟醚组（Iso组）和海藻糖组（Tre组）,每组20只。对照组小鼠不给予任何药物,将其置于持续通入2 L·min^-1氧气的麻醉箱中2h;Iso组和Tre组小鼠于麻醉前30 min分别经腹腔注射2 mL生理盐水或海藻糖（400 μg·kg^-1）稀释液,然后给予1.4%异氟醚吸入麻醉2 h。麻醉后6 h 取小鼠海马组织制备脑组织匀浆,应用 DCFH-DA荧光法检测小鼠海马组织中活性氧（ROS）水平;麻醉后24 h应用免疫组织化学法和Western blotting法检测小鼠海马组织中蛋白羰基化合物、硝基化酪氨酸和β-淀粉样蛋白（Aβ）_1-42蛋白表达水平,应用透射电镜检测海马神经元中蛋白聚集物的形成,应用TUNEL染色法观察小鼠海马组织中神经元凋亡率。结果：与对照组比较,Iso组小鼠海马组织中ROS水平、氧化蛋白羰基化合物、硝基化酪氨酸、Aβ_1-42蛋白表达水平和神经元凋亡率均明显增加（P < 0.05）;与Iso组比较,Tre组小鼠海马组织中ROS水平、氧化蛋白羰基化合物、硝基化酪氨酸、Aβ_1-42蛋白表达水平和神经元凋亡率均明显降低（P < 0.05）。结论：异氟醚能诱导转APP基因小鼠海马神经元蛋白质损伤和蛋白聚集,加剧氧化应激反应,增加脑内海马神经元凋亡,海藻糖能够拮抗异氟醚诱导的神经毒性。     

海藻糖联合葡萄糖负载红细胞后溶血效果

目的：探讨海藻糖和葡萄糖联合负载红细胞的效果,为冷冻干燥红细胞提供新的保存剂。方法：分别采用0、0.125、0.25、0.5和1 mol/L的海藻糖、葡萄糖以及海藻糖联合葡萄糖37℃负载红细胞6 h。然后采用邻甲苯胺法检测上清液中游离血红蛋白含量。结果：负载液浓度为1 mol/L时,3组的细胞溶血程度较重,即上清中游离血红蛋白浓度较高。在浓度低于1 mol/L时,海藻糖联合葡萄糖组与单独海藻糖负载组溶血程度没有明显差别,但是2者低于葡萄糖组。结论：在浓度小于1 mol/L时,海藻糖联合葡萄糖负载红细胞后对细胞的损伤较小,能够满足冻存的负载要求。     

海藻糖对低温保存的气管组织细胞活力的影响

目的探讨海藻糖对低温保存的气管组织细胞活力的影响。方法新鲜配制两组保存液，其中：Ⅰ组LPD DMSO，Ⅱ组LPD DMSO 海藻糖(0．10mol．L^-1)。切取SD大鼠气管后立即分别放入含上述两种溶液的冻存管，在程序降温仪降至-80℃后投入液氮中保存．分别在1，15，30，60，120d后对气管组织体外培养。并加入^3H—TdR做标记，检测细胞掺入率。结果采用^3H—TdR体外组织培养，低温保存后Ⅰ组气管^3H—TdR掺入率(88．72％～78．21％)明显高于Ⅱ组(80．99％～70．75％)，这种趋势可长时间持续(共120d)。结论含有海藻糖的LPD溶液在低温下对气管组织细胞有明显的保护作用；^3H—TdR体外组织培养的方法可以比较客观地反映了器官保存后的活力。