Upregulation of the GRIM-19 gene suppresses invasion and metastasis of human gastric cancer SGC-7901 cell line.

Gene associated with retinoid-IFN-induced mortality 19 (GRIM-19), as a novel IFN-beta/RA-inducible gene product, was identified as a potential tumor suppressor associated with growth inhibition and cell apoptosis. Recently, it has been reported that the apoptotic effects and apoptosis-related gene induction of GRIM-19 can be attenuated by GW112, indicating that GRIM-19 and GW112 are involved in a common signal transduction pathway. To investigate the signaling mechanisms that link GRIM-19 to GW112 and their functional role in tumor cell invasion and metastasis, we utilized adenovirus-mediated overexpression of GRIM-19 in the gastric cancer SGC-7901 cell line.

[Expression and identification of recombinant arresten in Pichia pastoris].

Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'.

[Preparation and identification of the monoclonal antibody against human endostatin].

Aim: To prepare monoclonal antibody against human endostatin for the in depth study on the mechanism of anti-tumor effect of endostatin.

Methods: Monoclonal antibody specific for endostatin was prepared using hybridoma technique and screened with ELISA. Ascites fluids were produced in BALB/c mice following in sequentical intraperitoneal injection of pristine and hybridoma cells.

Kinetic substrate quantification by fitting the enzyme reaction curve to the integrated Michaelis-Menten equation.

The reliability of kinetic substrate quantification by nonlinear fitting of the enzyme reaction curve to the integrated Michaelis-Menten equation was investigated by both simulation and preliminary experimentation. For simulation, product absorptivity epsilon was 3.00 mmol(-1) L cm(-1) and K(m) was 0.

Assay of Adenylyl Cyclase Activity by Ion-exchange High-performance Liquid Chromatography.

Adenylyl cyclase(AC)activity was determined by directly quantifying the product cAMP with ion-exchange HPLC. When AC reaction was terminated, the reaction mixture was kept at 0 degrees for 2 h to remove residual ATP utilizing the action of ATP-hydrolyzing enzymes existed in the crude AC preparation of membrane pellet. Papaverine was removed by the dichloromethane extraction, and cAMP was quantified by chromatography on a WAX-1 HPLC column with baseline-resolution.