^99mTc complex conjugated to insulin： CNS radiopharmaceuticals design based on principles of blood-brain barrier transport vector

Hydrophilic ^99mTc-EC and nonlipophilic ^99mTc- MAMA＇-BA complexes, owing to the existing of intact blood-brain barrier （BBB） in vivo, cannot cross from blood to brain. Previous studies showed that insulin is selectively transported by receptor-mediated transcytosis through the brain capillary endothelial wall that makes up the BBB. In this paper, based on the characteristic of the insulin receptor enriched in brain capillary, the complexes of hydrophilic ^99mTc-EC and nonlipophilic ^99mTc-MAMA＇-BA are conjugated to insulin respectively. After purification, the radiochemical purity of ^99mTc-EC-insulin and ^99mTc-MAMA＇-BA-insulin was 〉 90% and the stability in vitro was good. Expectation for the special formulation can be internalized and endocytosed into the capillary membrane by the vector-mediated brain delivery system, and transported ^99mTc-labeled conjugate through the BBB in vivo, thus enhancing brain uptake in mice. The biodistribution results of ^99mTc-EC-insulin and ^99mTc-MAMA＇-BA-insulin in mice in- dicated that the brain uptake was higher than ^99mTc-EC and ^99mTc-MAMA＇-BA to some extent. The ratios of brain uptake of ^99mTc-EC-insulin to ^99mTc-EC, ^99mTc-MAMA＇-BA-insulin to ^99mTc-MAMA＇-BA were 4-6 at 2 and 3 h post-injection respectively. In conclusion, the given results have illustrated a new way of brain uptake enhancing for nonlipophilic like complexes that have BBB delivery problems. It has a potential value for the ongoing development of ^99mTc-labeled radiopharmaceuticals for CNS receptors imaging.     

己糖激酶冻干粉的制备工艺及其酶学性质

为将发酵制备的液态己糖激酶制备成粉剂,应用于诊断试剂领域,对己糖激酶冻干保护剂进行了筛选优化;并对制得的己糖激酶干粉的酶学性质进行了研究。通过单因素试验、Plackett-Burman设计和最陡爬坡试验,以响应面分析确定其最优保护剂种类复配比例。结果表明:冻干保护剂选取蔗糖、乳糖和山梨醇效果最佳;且最优比为蔗糖4. 5%、乳糖2. 0%、山梨醇8. 0%,平均酶活保存率为78. 1%。己糖激酶干粉的最适反应温度为50℃;在45℃高温处理30 min后,仍具有60%的活力;最适反应p H为8. 0,在p H 6. 0～10. 0范围内具有较好的稳定性;为己糖激酶制剂在诊断试剂领域的应用奠定了基础。     

A method for determination of hexokinase activity by RP-HPLC

A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography(RP-HPLC).The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate.The separation degree of ATP and ADP was as good as 3.46(separation degree>1.5).In the range of 0.01-0.40 mmol/L,there was a good linear relationship between concentration of ADP and its peak areas(the correlation coefficient was 0.999).The relative standard deviation(RSD) of intraday was 1.43%-1.46%,that of interday was 2.12%-2.15%.At 30 °C(optimal temperature) and pH7.0(optimal pH),the recovery rate of hexokinase was 95.3%-97.6%.The results showed that the method had good precision and high recovery rate,and the enzyme activity was determined through only one-step reaction.To some extent,this method can reduce the cost.     

The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.     

Effects of vanadate on the activities of mice glucokinase and hexokinase

This study aimed at acquiring knowledge on the hypoglycemic mechanisms of sodium metavanadate (SMV) showed that the liver glucokinase and muscle hexokinase activities increased rapidly after oral SMV was given, and that the blood glucose level was correlated closely with the activities of the two enzymes but not with the insulin level; which indicated that SMV could improve the altered glucose phosphorylation in diabetic mice independently of stimulating insulin secretion. This was probably one of the mechanisms of hypoglycemic effects of SMV.