Resorufin-beta-O-glucuronide(KEG) is a substrate of the beta-glucuronidase enzyme which exhibited almost no fluorescence, upon addition of beta-glucuronidase a high fluorescent compound resorufin will be released. Resorufin, is a fluorescent compound possessing good optical properties. On the basis of the off-on type fluorescent reaction, REG can be served as the good fluorescent substrate of beta-glucuronidase. However, commercial available KEG is rather expensive due to the difficulty for preparation of REG and the studies on REG are very limited. This study aimed to adopt the mild biosynthesis approach to efficiently prepare REG, based on resorufin can be extensively metabolized to KEG by UDP-glucuronosyltransferases(UGTs). REG was prepared using resorufin as the starting material and liver microsomes from cynomolgus monkeys(CyLM) as the enzyme source. Resorufin(RE, 100 mu mol/L) was incubated in Tris-HCl(50 mmol/L, pH = 7.4, with 1% DMSO) with CyLM(0.5 mg/mL) at 37 degrees C for 4 h. After the optimization of the incubation conditions, the conversion of REG was more than 80%. A unique solid-phase extraction column(SPE) packed with C18WAX was used to enrich and purify the product KEG with high yield. The product was then identified by both LC-MS and NMR techniques. Finally, a high throughput screening method for beta-glucuronidase inhibitors was well established, based on the accurate kinetic parameters of REG towards beta-glucuronidase and the absorption and emission spectrum of REG.

Objective To study the expression of neuronal nitric oxide synthase (nNOS) in the renal cortex of type 1 diabetic rats. Methods The diabetic models were established by intraperitoneal injection of 50 mg/kg streptozotocin (STZ) once in rats. Rats were executed respectively at the first, second and fourth weeks after injection of STZ. Nitrite reductase was appllied to detect the content of nitrogen monoxide (NO), while immunohistochemical staining and Western blot were used to measure the expression of nNOS in renal cortex. Results Compared with normal control group, NO level in renal cortex was lower in diabetic model group at the first week (P < 0.05), higher at the second week (P < 0.05) and remarkably higher at the fourth week (P < 0.01).In renal cortex nNOS mainly expressed in maculer denser(MD) cells and in tubuls. The nNOS content was lower at the first week after injection of STZ(P < 0.05) , similar at the second week and higher at the fourth week in diabetic model group than in normal group respectively . Conclusion The reduced expressions of nNOS in renal cortex maybe contribute to the decrease of NO in type 1 diabetic rats .

Aim: To observe the expression of neuronal nitric oxide synthase (nNOS) in the renal tissues of diabetic rats in the early period. Methods: The experiment was conducted in the Center of Science and Experiment and the Department of Histology and Embryology, Jinzhou Medical College between March and October 2005. Totally 60 rats of 2-3 months old were randomly divided into diabetic model group and normal control group, with 30 rats in each group. After 12-hour fasting, the rats in diabetic model group were used to make diabetic models by intraperitoneal injection of 50 mg/kg streptozotocin (STZ) once, while rats in normal control group were given citric acid buffer of the same volume. Rats were executed respectively at the first, second and fourth weeks to fetch renal tissues. Nitrite reductase was applied to detect the content of nitrogen monoxide (NO), while immunohistochemical staining was used to measure the expression of nNOS in macula densa (MD) cells. The gray values of nNOS positive neurons in MD were assayed with Mivnt cell image analysis system. Results: Totally 60 rats entered the result analysis without death. 1 The nNOS content of rats in diabetic model group was obviously lower than that of normal control group at the first week, with the significant difference [(1 068.01±61.61), (2 329.99±200.62) mmol/L, P < 0.05], but gradually increased at the second and fourth weeks in diabetic model group. There was insignificant difference at the second week [(2 334.59±192.40), (2 372.11±193.34) mmol/L, P > 0.05], whereas significant difference at the fourth week [(4 939.51±133.44), (2 407.30±174.94) mmol/L, P < 0.05] between two groups. 2 Renal cortex NO level was lower in diabetic model group than in normal control group at the first week, with the significant difference [(8.56±1.25), (11.23±2.08) μmol /L, P < 0.05], higher than that of normal control group at the second week, with the significant difference [(13.71±1.86), (9.93±2.01) μmol/L, P < 0.05], and remarkably higher than that of normal control group at the fourth week, with the remarkably significant difference [(16.29±2.46), (10.99±1.64) μmol /L, P < 0.01]. 3 The blood sugar of rats were higher in diabetic model group than in normal control group at three time points, with the significant difference (P < 0.01). Conclusion: The reduced expressions of nNOS in renal tissues maybe contribute to the decrease of NO in diabetic rats with renal disease of early course.