Determination of Acid β-Galactosidase Activity: Methodology and Perspectives

Early, accurate diagnosis of lysosomal storage disorders is a major challenge, even for trained specialists. Finding innovative, accurate diagnostic methods, and high throughput, cost-effective tools are crucial to medical progress and will contribute to improved quality of life. The goal of this work was to improve currently used protocols to determine activity of acid β-galactosidase, and discuss the possibility analysing lysosomal enzymes with microfluidic systems. A principle of the determination of β-galactosidase activity was fluorometric measurement of a deprotonated form of 4-methylumbelliferone released in the enzymatic reaction. Measurements were performed using Jurkat T cells as a source of the enzyme. We observed the temperature-dependent substrate inhibition effect and determined the substrate (4-MU-β-d-galactopyranoside) concentration which should be used to determine acid β-galactosidase activity at 37 °C (0.8 mM) and at room temperature (0.6 mM). We proved that the sample incubation time may be significantly reduced to only a few minutes. We also showed that the amount of alkaline buffer used to stop the enzymatic reaction may be minimized and even, in some cases, eliminated. The presented results show how the sensitivity of the available methods to diagnose patients suffer from gangliosidosis GM1 or Morquio B disease can be improved. The proposed method may be easily implemented with microfluidic systems, which currently are promising tools for point-of-care applications.     

以特色打造高职教育品牌

在我国,高等职业技术教育是一种新兴的高等教育类型。在激烈竞争中,要办好高职教育,必须以特色取胜。而要打造高职教育特色品牌,必须充分认识其本质特征,更新办学观念。要大力推行“六双”改革,按职业岗位（群）设置专业,按照“厚基础,宽口径,强技能”的原则建立基础知识、专业理论和职业技能三大“平台”, 建设“理论型”、“技能型”、“双师型”并重的师资队伍和产教研有机结合的办学机制。     

基于Cyt b基因的雀形目15种鸟类系统发生关系初探

以线粒体DNA的Cyt b基因全序列作为分子标记,对雀形目(Passeriformes)6科15种鸟类的分类地位及系统发生关系进行了分析.结果显示:全序列中含有变异位点454个,简约信息位点337个.采用邻接法、最大简约法、最大似然法和贝叶斯法分别构建了系统发生树及其合一树,结果表明:黄鹂科(Oriolidae)首先分化出来,接着依次分化出卷尾科(Docruridae)、鸦科(Corvidae)、伯劳科(Laniidae)、河乌科(Cinclidae)和椋鸟科(Sturnidae)鸟类;这6科鸟类构成一个单系群,在这一单系群里椋鸟科是最晚分化出的类群.     

小灵猫红细胞免疫功能的研究

本文采用免疫花环试验法测定了健康小灵猫红细胞上C_(3b)受体和免疫复合物的数量33例。红细胞上C_(3b)受体花环率的均值为9.21±1.81;免疫复合物花环率的均值为5.50±1.36。这一研究结果充平表明,小灵猫的红细胞不但具有携氧、运输气体等功能外,而且还具有重要的免疫功能。     

Effect of streptozotocin-induced diabetes on oxidative energy metabolism in rat liver mitochondria—A comparative study of early and late effects

The reports in the literature on effects of diabetes on mitochondrial energy-linked functions are conflicting. Hence we carried out systematic studies to evaluate the effects at the early and the late stages of the disease using STZ-diabetic rat as a model. At the end of one week, after induction of diabetes, respiration rates with glutamate and succinate as the substrates increased; respiration rates with other substrates e.g. β-hydroxybutyrate, pyruvate + malate and ascorbate + TMPD were not affected despite substantial decrease in the β-hydroxybutyrate dehydrogenase activity and cytochrome b and c+c₁ contents. Insulin treatment brought about increase in the cytochrome contents beyond control values. The ATPase activity was generally low in the diabetic animals and was not restored by insulin treatment. At the end of one month, the respiratory activities with all the substrates were generally low. Insulin treatment either restored or stimulated the respiration rates beyond control values. The content of cytochromes was differentially affected in the diabetic animals, but insulin treatment caused significant increase beyond control levels. The pattern for ATPase activity was similar to the early effects. At both the stages i.e. early and late stages of diabetes the mitochondria were tightly coupled. The ADP/O ratios were in normal expected ranges and the respiratory control ratios were comparable with the control groups. Insulin treatment resulted in apparent restoration of respiratory activity. However, the effects on the cytochromes and dehydrogenases activities were differential. Taken together the two observations would suggest that the mitochondria were not re-instated to normality despite apparent restoration of respiratory function.     

天然维生素E对药物代谢影响的研究进展

最近研究表明天然维生素E能激活PXR受体,调控其下游靶基因CYP3A及MDR1的表达.而临床上50%的药物是通过CYP3A代谢,P-糖蛋白能转运大部分的抗生素,抗疟药,肿瘤化疗药物,CYP3A与P-糖蛋白底物及调控机制均相似,两者共同抵御外界化学物质进入细胞.在药物联合使用中天然维生素E很有可能影响其它药物代谢,产生药物相互作用.由P-糖蛋白或CYP3A代谢的药物与天然维生素E同时服用,可能会因为天然维生素E影响了CYP3A或P-糖蛋白的活性而产生药物相互作用.导致不良反应.     

Kinetic analysis of γ-glutamyltransferase reaction process for measuring activity via an integration strategy at low concentrations of γ-glutamyl p-nitroaniline

At 0.12 mmol/L γ-glutamyl p-nitroaniline (GGPNA), an improved integrated method was developed for kinetic analysis of γ-glutamyltransferase (GGT) reaction process and the integration with the classical initial rate method to measure serum GGT. For the improved integrated method, an integrated rate equation, which used the predictor variable of reaction time and considered inhibitions by both GGPNA and products, was nonlinearly fit to GGT reaction processes. For the integration strategy, classical initial rates were estimated when GGPNA consumption percentages were below 50%; otherwise, maximal reaction rates of GGT were estimated by the improved integrated method and converted into initial rates according to the differential rate equation at 0.11 mmol/L GGPNA. The integration strategy was validated using optimized GGT kinetic parameters and 10-s intervals to record reaction curves within 8.0 min. By the integration strategy, there was a linear response from 0.9 to 32.0 U/L GGT, coefficients of variation were below 3.5% for GGT from 8.0 to 32.0 U/L (n=5), and GGT activities in clinical sera responded linearly to their classical initial rates at 2.00 mmol/L GGPNA with an expected slope. Therefore, the integration strategy was successful in measuring GGT at 0.12 mmol/L GGPNA.     

走向人文化的教育——新世纪我国教育的价值取向

在社会转型的特殊条件下,我国的教育要很好地完成自己的使命,就必须做到走进生活———建基教育的基础;关注生命———坚持教育的本真;提升精神———实现教育的追求;重塑信仰———恪守教育的灵魂。     

基于班级作文博客的作文诊断性评价研究

班级作文博客在当前作文教学中逐渐得到广泛应用。相对传统的教学手段,利用班级作文博客对作文进行诊断性评价具有不少优势。当前,运用博客对作文进行的诊断性评价还存在不少问题。班级博客诊断评价作文应在坚持发展、自主、全面性的基本原则下综合运用多种方法进行评价。     

管蚜蝇族7种食蚜蝇Cty b基因序列差异及系统进化关系

采用PCR和测序方法,首次获得管蚜蝇族(Eristalini)7种食蚜蝇线粒体基因组的Ctyb基因部分序列(419bp).经对位排列,序列间未见有插入和缺失,共检测到119个可变核苷酸位点,变异率为28 40%,其中简约位点74个.属内序列差异值在5 01%～9 55%之间,属间序列差异值在10 02%～16 23%之间.以直翅目的中华稻蝗为外群,用多种方法构建进化关系树,结果类似.从4个属间关系来看,管蚜蝇属和斑眼蚜蝇属首先聚合为姊妹群,BCL值为61%,其次与条胸蚜蝇属聚合,BCL值为89%,宽盾蚜蝇属最后聚合.管蚜蝇族的4个属的聚类系统发生树为单系.