The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC).The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.
A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen‐dependent hydroxylation of (S)‐naringenin [(S)‐5, 7, 4′‐trihydroxyflavanone] to eriodictyol (5, 7, 3′, 4′‐tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3′‐hydroxylase reaction. In contrast prunin (naringenin 7‐O‐β‐glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4‐hydroxylase, did not catalyse hydroxylation of 4‐coumaric acid to caffeic acid.
3′‐Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P‐450‐dependent flavonoid 3′‐monooxygenase. Pronounced differences in the inhibition of flavonoid 3′‐hydroxylase and cinnamate 4‐hydroxylase were found with EDTA, potassium cyanide and N‐ethylmaleimide.
Irradiation of the cell cultures led to increase of flavonoid 3′‐hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light‐induction of phenylalanine ammonialyase and cinnamate 4‐hydroxylase.
In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3′‐hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia‐lyase, cinnamate 4‐hydroxylase, and NADPH‐cytochrome reductase. The results prove that flavonoid 3′‐hydroxylase and cinnamate 4‐hydroxylase are two different microsomal monooxygenases.
A microsomal preparation from elicitor-challenged soybean cell suspension cultures catalyzes an NADPHdependent and dioxygen-ldependent 6a-hydroxylation of 3,9-dihydroxypterocarpan to 3,6a,9-trihydroxypterocarpan. The latter is a precursor for the soybean phytoalexin glyceollin. No reaction is observed with NADH. The 6a-hydroxylase is inhibited by cytochrome c.Optical rotatory dispersion spectra of the enzymatic product formed from racemic dihydroxypterocarpan and of the remaining unreacted substrate proved that the product has the natural (6aS, 1laS)-configuration and that hydroxylation proceeds with retention of configuration.The 6a-hydroxylase was also found in elicitor-challenged soybean seedlings. The results indicate that the 6a-hydroxylase is specifically involved in the biosynthesis of glyceollin.
The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2-DE and MALDI-MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.-L., et al., Clin. Cancer Res. 2005, 11, 6550-6557).Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano-flow 2-D-LC-ESI-MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2-DE/MALDI-MS approach, whereas 232 proteins were unique to the 2-D-LC-ESI-MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2-D-LC-ESI-MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2-DE/MALDI-MS approaches and has the potential to identify novel biomarkers.
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