Modeling of alphak/gamma2 (k=1, 2, 3 and 5) interface of GABA A receptor and docking studies with zolpidem: implications for selectivity.

The three-dimensional models of the alphak/gamma2 (k=1, 2, 3 and 5) interface of GABA(A) receptors, which included the agonist-binding site, were constructed and validated by molecular modeling technology. To investigate the mechanism of alpha subunit selectivity of zolpidem, docking calculations were used to illustrate the potential binding modes of zolpidem with different alpha subtypes. The results revealed that there were three reasons resulting in the distinct binding affinity of zolpidem to different alpha subtype. Firstly, the number of hydrogen bonds of agonist-receptor complex would determine the magnitude of binding affinity. Secondly, the His residue in loop A of alpha subunit was indicated as a key role of benzodiazepine binding. Thirdly, the side chain of Glu in loop C reduced the affinity of zolpidem to those receptors containing alpha2, alpha3 or alpha5 subunits.

Expression of a wheat cytochrome P450 monooxygenase in yeast and its inhibition by glyphosate.

Glyphosate is a non-selective herbicide which acts by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase. Wheat cytochrome P450 monooxygenase specifically catalyzes the metabolism of some sulfonylurea herbicides such as chlorsulfuron and triasulfuron. Here we report that glyphosate is an inhibitor of a wheat cytochrome (CYP71C6v1), the cDNA of which was amplified by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain had a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum, which is a typical spectral characteristic. The addition of glyphosate to the microsomal protein resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand. A spectral dissociation constant, K(s) of 70 micromol litre(-1) was observed and an IC50 of 11 micromol litre(-1) was found for glyphosate inhibition of CYP71C6v1 P450 activity.

[Establishment and application of the model of islet impaired by NO free radical released from streptozotocin].

For diabetes mellitus, little research has been done on the tissue-based or cell-based drug screening model, which has advantages over traditional animal diabetic model in high specificity, high screening volume, low cost and simple manipulation. Considering that the maintenance of complete islet tissue structure is the prerequisite for islet cells to perform their functions normally, an in vitro islet-based drug screening model for diabetes mellitus was established and evaluated. Pancreatic islets were isolated from 3 weeks old mice of either sex by collagenase digestion and density gradient centrifugation as prescribed by Ramanadham S. The volume of 0.1% (W/V) collagenase IV, 0.1% (W/V) Hyaluroridase and 0.1% (W/V) DNase I were 4 times, 2 times and 1 times that of the islets to be digested. And a 2 hours' cold digestion at 4 degrees C was followed by a 10 minutes' warm digestion at 37 degrees C. Under the optimized digestion condition, the islet recovery could be increased by 10%. The isolated islets could survive 6 weeks in vitro and show stable insulin secretion in the first 10 days after inoculation. The obtained islets were cultured in RPMI-1640 medium at 37 degrees C with 5% CO2. Then a diabetic model was established by selecting streptozotocin (STZ) as the evocator and nitric oxide (NO) as the responding index. After 1 day's inoculation, islets culture was treated with STZ, whose concentration ranged from 0 to 5.0 mmol/L. NO was measured by a colorimetric assay at 540nm based on the Griess reaction for 10 min with 0.1 mL Griess reagent and 0.1 mL culture supernatants. Insulin secretion was assayed by RIA methods. Due to the islets-related inoculation variations, NO release and insulin content were both expressed as a percentage of the value recorded in basal experiment which was in the only presence of Krebs culture medium. It was testified that the amount of NO released from islet itself remained steady at 30-35 mmol/L regardless of the changes of STZ concentration from 0 to 5.0 mmol/L. However the NO content in the supernatants of islets culture had close relationship with STZ concentration. This indicated that in this STZ-induced islet diabetic model, NO mainly comes from STZ when it dissolves in water. On the other hand, when STZ changed from 0 to 5.0 mmol/L, the dose-dependent relationship between NO content and insulin secretion showed that the increase of NO came along with the decrease of insulin secretion, which is an important symbol of islet function. As a kind of oxidative free radical, NO is capable of impair islet cells. Thus, NO is a reliable responding index of the model. The optimal STZ concentration in the model is finally determined to be 5.0 mmol/L, under which condition the NO content and insulin secretion is 10.81 times and 0.43 times that in the medium before STZ is added. So if anything is effective in lowering the NO content in the culture, it could protect islets cells from the oxidative attacks of NO. Finally, as an application of the model, the scavenging effect of KOSCr on NO was studied. In a series of KOSCr with different chromium content, all had shown better NO scavenging effects than KOS itself, which could give us an enlightenment of the influence of chromium ion on oligosaccharide. And 1 g/mL KOSCr with 3.519% chromium content can significantly inhibit the NO formation. This has lain a theoretic basis for the research of KOSCr bioactivity and quality control. These results suggested that the STZ-induced diabetic islet model which is impaired by NO free radical can be used effectively, fast and conveniently when screening potential diabetes drugs.