Purification and partial characterization of thioredoxin reductase from the hepatopancreas of the mollusc Mytilus galloprovincialis Lam.

Thioredoxin reductase (TrxR, EC 1.6.4.5) is a ubiquitous flavoenzyme that is present from Archaea to humans, and it is the only enzyme capable of catalyzing the reduction of thioredoxin (Trx) by nicotinamide adenine dinucleotide phosphate (NADPH). Although TrxR has been purified and characterized from different bacteria, plants, and mammalian organisms, a survey of the literature revealed no studies on the purification and characterization of TrxR from the mussel Mytilus galloprovincialis Lam. In this study, TrxR was purified to homogeneity from the hepatopancreatic tissue of M. galloprovincialis Lam. by extraction, ammonium sulfate precipitation, and DEAE-Sepharose CL-6B anion and 2', 5'-ADP-agarose chromatographies, and some of its kinetic properties were examined. Molar mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed only a single protein band corresponding to a molecular weight of 35 kDa. Optimum pH and temperature were found to be 7.0 and 60 degrees C, respectively. Km and Vmax values for NADPH were found to be 85 mu mol and 4.82 mu mol/min/mg, respectively. For 5,5'-dithiobis (2-nitrobenzoic) acid (DTNB), the Km and Vmax values were 193 mu mol and 1.32 mu mol/min/mg, respectively. Increasing the knowledge on the kinetic properties of TrxR will significantly increase the prospects of enzyme application as an oxidative stress biomarker in mussels and fishes for monitoring contamination in coastal environments

Identification of AKT1/CTNNB1 mutations conferring cetuximab and chemotherapeutic drugs resistance in colorectal cancer treatment

In anti-cancer therapy, the effectiveness of therapeutics is limited by mutations causing drug resistance.1KRASmutations are the only determinant for cetuximab resistance in colorectal cancer (CRC) patients.2Therefore, it is very important to determine new predictive mutations in CRC treatment.3In this study, the association of AKT1/CTNNB1mutations with the drug resistance against cetuximab and some chemotherapeutics used in the CRC treatment were investigatedin vitroby using site-directed mutagenesis, transfection, Western Blot methods, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell growth inhibition assay. The cetuximab resistance was higher in the presence of AKT1 E17K, E49K, and L52R mutations and, CTNNB1 T41A, S45F, and S33P mutations compared to that of the WT. AKT1/CTNNB1mutations were also determined to be associated with oxaliplatin, irinotecan, SN-38, and 5-fluorouracil resistance. Furthermore, mutant cell viability in oxaliplatin treatment was more effectively inhibited than that of the other drugs. These findings provide evidence that CRC patients carryingAKT1/CTNNB1mutations may have resistance to cetuximab and frequently used chemotherapeutics, and these mutations may serve as an important predictive biomarker which responsible for drug resistance