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.

<p>Hydrogen peroxide (H₂O₂), a reactive oxygen specie (ROS), is most commonly associated with oxidative stress causing cytotoxic effects on living cells. Oxidative stress has been implicated in various conditions including neurodegenerative diseases, autoimmune diseases and cancer. In addition H₂O₂is produced as a defense mechanism against pathogens, as being released by activated phagocytes.<em> </em>In recent years, H₂O₂ has become established as an important regulator of signal transduction in eukaryotic cells. Hydrogen peroxide is generated both intracellularly and extracellularly in response to various stimuli including cytokines and growth factors. There are different mechanisms by which H₂O₂ is generated, facilitating signal transduction in cells; through NOX-system in miyochondria, via singlet oxygen, receptor/ligand interaction or by redox active metal ions. The HIV glycoprotein 120 (gp120) is associated with HIV dementia and it is known as a neurotoxin that causes neuronal damage. It has been proposed that free radicals may be involved in the pathogenesis caused by gp120. In addition the truncated form of thioredoxin (Trx80) is known to stimulate HIV replication in HIV infected cells, however, the exact mechanism is not known. A possible way both proteins may mediate their activity is by inducing H₂O₂ production. The aim of this study was to investigate H₂O₂ production induced by the proteins gp120 and Trx80. In order to detect H₂O₂ production an assay based on the fluorescent compound Amplex Red, was established. The assay was used to detect H₂O₂ released by gp120 and Trx80 in a cell-free environment, in a cell-system and in the presence of metal ions (copper ions) with a physiological reductant (ascorbate). We did not detect H₂O₂ production induced by gp120 and Trx80 respectively, using our assay, however, other ROS such as hydroxyl radicals may have been generated although they were not detectable with our method. Hence, further studies are needed in order to fully understand how gp120 and Trx80 mediate their activity.</p>

The first study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe3O4 nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H2O2, the reaction pH between Fe3O4 NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe3O4 NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe3O4 NPs and oxidase for the quantification of specific substrates through the H2O2-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 £gM using glucose oxidase-Fe3O4, galactose oxidase-Fe3O4, and choline oxidase-Fe3O4 composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. The second study follows the first one. Using the catalytic activity of Fe3O4 NPs with AU to detect four kinds of neurotransmitter, such as dopamine, L-DOPA, adrenaline (epinephrine) and noradrenaline (norepinephrine). Because of there is specific interaction between Fe3O4 NPs and catecholamines (CAs), the Fe3O4 NPs will form CAs-Fe3O4 NPs composites in presence of CAs. The CAs on the Fe3O4 NPs surface must shelter the reaction between AU and H2O2, cause the fluorescence to be turned-off. The CAs just like a inhibitor, to inhibit the catalytic activity of Fe3O4 NPs. Therefore, we could use this inhibited system to detect the CAs compound concentration in the real sample.

Hydrogen peroxide (H2O2), a reactive oxygen specie (ROS), is most commonly associated with oxidative stress causing cytotoxic effects on living cells. Oxidative stress has been implicated in various conditions including neurodegenerative diseases, autoimmune diseases and cancer. In addition H2O2 is produced as a defense mechanism against pathogens, as being released by activated phagocytes. In recent years, H2O2 has become established as an important regulator of signal transduction in eukaryotic cells. Hydrogen peroxide is generated both intracellularly and extracellularly in response to various stimuli including cytokines and growth factors. There are different mechanisms by which H2O2 is generated, facilitating signal transduction in cells; through NOX-system in miyochondria, via singlet oxygen, receptor/ligand interaction or by redox active metal ions. The HIV glycoprotein 120 (gp120) is associated with HIV dementia and it is known as a neurotoxin that causes neuronal damage. It has been proposed that free radicals may be involved in the pathogenesis caused by gp120. In addition the truncated form of thioredoxin (Trx80) is known to stimulate HIV replication in HIV infected cells, however, the exact mechanism is not known. A possible way both proteins may mediate their activity is by inducing H2O2 production. The aim of this study was to investigate H2O2 production induced by the proteins gp120 and Trx80. In order to detect H2O2 production an assay based on the fluorescent compound Amplex Red, was established. The assay was used to detect H2O2 released by gp120 and Trx80 in a cell-free environment, in a cell-system and in the presence of metal ions (copper ions) with a physiological reductant (ascorbate). We did not detect H2O2 production induced by gp120 and Trx80 respectively, using our assay, however, other ROS such as hydroxyl radicals may have been generated although they were not detectable with our method. Hence, further studies are needed in order to fully understand how gp120 and Trx80 mediate their activity.

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.