Stable Transfection of the Singlet Oxygen Photosensitizing Protein SOPP3: Examining Aspects of Intracellular Behavior.

Protein-encased chromophores that photosensitize the production of reactive oxygen species, ROS, have been the center of recent activity in studies of oxidative stress. One potential attribute of such systems is that the local environment surrounding the chromophore, and that determines the chromophore's photophysics, ideally remains constant and independent of the global environment into which the system is placed. Therefore, a protein-encased sensitizer localized in the mitochondria would arguably have the same photophysics as that protein-encased sensitizer at the plasma membrane, for example. One thus obtains a useful tool to study processes modulated by spatially localized ROS. One ROS of interest is singlet oxygen, O2 (a1 Δg ). We recently developed a singlet oxygen photosensitizing protein, SOPP, in which flavin mononucleotide, FMN, is encased in a re-engineered light-oxygen-voltage protein. One goal was to ascertain how a version of this system, SOPP3, which selectively makes O2 (a1 Δg ), in vitro, behaves in a cell. We now demonstrate that SOPP3 undergoes exacerbated irradiation-mediated bleaching when expressed at either the plasma membrane or mitochondria in stable cell lines. We find that the environment around the SOPP3 system affects the bleaching rate, which argues against one of the key suppositions in support of a protein-encased chromophore.

A Fragment of Collagen Type VI alpha-3 chain is Elevated in Serum from Patients with Gastrointestinal Disorders.

Extracellular matrix (ECM) remodeling is a hallmark of the pathology of gastrointestinal disorders. Collagen type VI (COL6) is produced by fibroblasts, and the COL6 α3-chain has shown to be elevated in patients with ulcerative colitis (UC), Crohn's disease (CD) and colorectal cancer (CRC). Measuring COL6α3 in serum may therefore have potential as a biomarker for gastrointestinal disorders. The aims of this study were to develop and validate a competitive ELISA targeting a specific neo-epitope of COL6α3 and evaluate its associations with the gastrointestinal disorders UC, CD and CRC, in comparison to healthy controls. A monoclonal antibody was raised against a matrix metalloproteinase-2 and -9 specific cleavage site of COL6α3 (C6Mα3) and employed in a competitive enzyme-linked immunosorbent assay (ELISA). The assay was developed and technically validated. Levels of C6Mα3 were measured in serum from patients with UC (n = 58), CD (n = 44) and CRC (n = 39) and compared to healthy controls (n = 32). The levels of C6Mα3 were elevated in patients with UC, CD and CRC patients compared to healthy controls (all p < 0.0001). The area under the receiver operating characteristics (AUROC) curve for separation of patients with UC from healthy controls was 0.972 (95% CI: 0.925-1.020, p < 0.0001), with CD from healthy controls was 0.947 (95% CI: 0.885-1.009, p < 0.0001) and with CRC from healthy controls was 0.890 (95% CI: 0.809-0.972, p < 0.0001). We developed a technically robust assay targeting a fragment of COL6, which was elevated in serum from patients with UC, CD and CRC.