Lysyl oxidase (LOX) and the four lysyl oxidase like proteins, LOXL, LOXL2, LOXL3 and LOXL4, are copper-containing amine oxidases constitute a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes, catalyzing the cross-linking of extracellular matrix (ECM) proteins. LOXL2 induces epithelial-to-mesenchymal transition (EMT), which is associated with hypoxia, enhanced invasion, cancer metastasis and poorer cancer prognosis. Furthermore, upregulation of LOXL2 mRNA and/ or protein levels has been detected in undifferentiated breast, colon, esophagus and larynx carcinomas. The aim here is to create and optimize a method to produce large yields of enzymatically active recombinant LOXL2 protein from mammalian cells. Two viral transductions systems were used to transfect CHO-K1 cells to overexpress LOXL2 protein. Comparing lentivirus transduction with adenovirus transduction, it was found that adenovirus transduction expressed 2.18 fold the amount of enzymatically active LOXL2 compared to lentivirus transduction (P<0.05). The average LOXL2 yield of lentivirus and adenovirus transduction systems as calculated by BCA assay was 184.5 µg and 403 µg, respectively. The average specific LOXL2 enzymatic activity were calculated using an Amplex red assay and found to be 0.443 and 0.444 nmol/μg of LOXL2 in 30 minutes in lentivirus and adenovirus methods, respectively, with no statistically significant difference (P>0.05). Expression and purification of LOXL2 were confirmed by SDS-PAGE and Western blot. Optimizing this method to purify large yields of LOXL2 is a practical aid in revealing the exact structure and function of the LOX family of proteins.

Parkinson's disease (PD) is a neurodegenerative condition that affects millions of people worldwide. The exact etiology of PD is unknown. However, it is well established that environmental factors contribute to the onset of PD. In particular, chemicals such as the insecticide Rotenone have been shown to increase the death of dopaminergic (DA) neurons by increasing levels of reactive oxygen species (ROS). ROS such as hydrogen peroxide (H2O2) have been shown to be elevated above basal levels in PD patients. Currently, to measure H2O2 concentrations, a commercially available (Amplex® Red) fluorescent assay is used. However, the assay has limitations: it is not completely specific to hydrogen peroxide and can only measure extracellular ROS concentrations. This research focuses on testing an electrochemical sensor that uses cyclic voltammetry to quantitatively determine concentrations of H2O2 released from a cell culture. The sensor was first tested in normal cell culture conditions. Next, chemical interference was reduced and the sensor was optimized for accuracy by altering protein concentrations in the media. Finally, Rotenone was added to a cell culture to induce H2O2 production. Near real-time measurements of H2O2 were taken using the sensor and comparisons made to the fluorescent assay method. Overall, we are trying to determine if the electrochemical sensor can selectively and quantitatively measure H2O2 released from cells. Being able to track the production, migration and concentration of H2O2 in a cell can help researchers better understand its mechanism of action in cell death and oxidative damage, thus getting closer to finding a cure for PD.