Purification and extraction of fibroblast growth factor 21 (FGF-21) protein by sumo fusion in Escherichia coli.

Fibroblast growth factor 21 has recently discovered its pivotal role in glucose, lipid metabolism and regulation of energy homeostasis. Further, it has helped in forming great strides for treatment of chronic diseases like diabetes and inflammation. FGF-21 was sub-cloned into the SUMO vector and was induced for expression in Escherichia coli Rosetta. The recombinant plasmid was transformed into Escherichia coli strain. FGF-21 was induced by IPTG and purified by Ni-NTA agarose (Nickel-nitrilotriacetic acid) column. The purified fusion protein was cleavaged by SUMO protease I to obtain recombinant FGF-21 with high purity. The purified protein was tested for its biological activity of FGF-21. HepG2 cell model was used to detect the regulation of glucose uptake activity of FGF-21 and were further treated with different concentrations of FGF-21.The residual glucose content in medium was measured using the glucose oxidase-peroxidase method. The results indicated that FGF-21 protein had a role in regulating the glucose uptake on HepG2 cells and the effect was significantly dose-dependent manner. In order to further verify whether purified FGF-21 protein obtained has biological activity in diabetic model. Studies have demonstrated that FGF-21 had a greater efficacy in dropping blood glucose in streptozotocin induced diabetic mice.

Eugenol and liposome-based nanocarriers loaded with eugenol protect against anxiolytic disorder via down regulation of neurokinin-1 receptors in mice.

Anxiety disorder is a psychiatric disorder characterized by extreme fear or worry. It is highly prevalent worldwide which affects daily life and is also an enormous health burden. Neurokinin 1 receptor (NK1R) is a G protein coupled receptor, expressed in both central and peripheral nervous system, involved in affective behaviors. NK1R has established role in anxiety and it is also an important target for pathogenesis of anxiety disorder. Therefore, it has been hypothesized in previous studies that the blockades of NK1R may have antidepressant and anxiolytic effects. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of NK1R was analyzed by real time RT-PCR. Moreover, the NK1R protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating NK1R protein expression in mice.

Glutathione, Cysteine, and D-Penicillamine Role in Exchange of Silver Metal from the Albumin Metal Complex.

The purpose of this study is to investigate the exchange reaction taking place among the bovine serum albumin (BSA), 5,5'-dithiobis-(2-nitrobenzoic acid (ESSE), reduced glutathione, N-acetylcysteine, D-penicillamine (thiolates), and silver metal (AgI). For this purpose, stock solutions of BSA and Ellman's reagent were prepared by dissolving 264 mg of BSA in 5 ml of reaction buffer (0.1 M KH2PO4 at pH 7.8) and 23.8 mg of ESSE in 1.0 ml of reaction buffer which were mixed together. Mixture of BSA-AgI was prepared in a separate procedure by dissolving 0.17 mg of silver nitrate in 1 ml of reaction buffer and then dissolving BSA (200 mg) in the same solution of silver nitrate. Blocking of Cys-34 of BSA with AgI was confirmed by treating different dilutions of BSA-AgI (500 µM) solutions with the solutions of ESSE (85 µM) and ES- (85 µM) and recording the spectra (300-450) with a UV-visible spectrophotometer. The chromatographed AgI-modified BSA ((BSA-S)AgI)) samples (typically 500 µM) were subsequently mixed with thiolates (reduced glutathione, N-acetylcysteine, and D-penicillamine). AgI and modified BSA (typically 500 µM each) were treated with these low molecular weight thiolates and allowed to react overnight followed by chromatographic separation (Sephadex G25). The redox reactions of AgI-modified BSA with various low molecular weight thiols revealed a mechanically important phenomenon. In the case of reduced glutathione and N-acetylcysteine, we observed the rapid release of a commensurate amount of Ellman's anion, indicating that an exchange has taken place and low molecular weight thiols (RSH) substituted AgI species at the Cys-34 of BSA eventually forming disulfide (BSA-SSR) at Cys-34. It can be anticipated from the phase of study involving bovine serum albumin that low molecular weight thiolates (reduced glutathione and N-acetylcysteine) take off AgI which are attached to proteins elsewhere in the physiological system, making these toxic metals free for toxic action.