Differential Regulation of Proteins and a Possible Role for Manganese Superoxide Dismutase in Bioluminescence of Panellus stipticus Revealed by Suppression Subtractive Hybridization

Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.

Mitochondrial DNA4977-bp deletion correlated with reactive oxygen species production and manganese superoxide dismutase expression in gastric tumor cells

Background Mitochondrial DNA 4977-bp deletion （△mtDNA^4977） was reported in many human neoplasia. However, its biological significance remains to be evaluated and the molecular mechanism needs to be investigated. In this study, we analyzed the frequency of △mtDNA^4977 in gastric cancer （GC） cell lines and tissues, as well as reactive oxygen species （ROS） contents and manganese superoxide dismutase （MnSOD） expression levels in GC cell lines to explore its biological significance and molecular mechanism. Methods Semi-quantitative PCR and real-time PCR were used to detect the incidence of △mtDNA^4977 in 13 GC cell lines and 272 human gastric tissues （108 GC specimens and the respective adjacent normal tissues, and 56 normal gastric mucosa from non-cancer patients）. We further identified intracellular ROS production by flow cytometry and MnSOD expression by semi-quantitative reverse transcription-PCR （RT-PCR） and Western blotting. Statistical analyses were carried out using the Logistic regression analysis and Kaplan-Meier method. Results Based on our earlier study, we optimized the PCR amplification condition by reducing the cycle number. In this study, we systematically documented the high incidence of △mtDNA^4977 in GC cell lines （10/13, 76.9%）, GC tissues （86/108, 79.6%）, matched normal tissues （73/108, 67.6%）, and normal gastric mucosa of non-cancer patients （29/56, 51.8%）. A significantly higher incidence of mutated △mtDNA^4977 was observed in GC tissues with respect to the adjacent normal tissues （79.6% vs 67.6%, P=-0.045）, and they were both higher than that in normal controls （P 〈0.05）. Most importantly, we linked the △mtDNA^4977 mutations with the expression level of MnSOD and ROS contents. The cell lines containing lower expression level of MnSOD was found to have generally higher frequent △mtDNA^4977 and more ROS. Conclusion The decreased anti-oxidative ability, which leads to increased ROS contents, is correlated with the mtDNA damage during gastric carcinogenesis.

The Expression of Manganese Superoxide Dismutase Gene from Nelumbo nucifera Responds Strongly to Chilling and Oxidative Stresses

A manganese superoxide dismutase （Mn-SOD） gene, NnMSD1, was identified from embryonic axes of the sacred lotus （Nelumbo nucifera Gaertn.）. The NnMSD1 protein contains all conserved residues of the Mn-SOD protein family, including four consensus metal binding domains and a signal peptide for mitochondrial targeting. Southern blot analysis suggests the existence of two Mn.SOD genes in sacred lotus. NnMSD1 was highly expressed in developing embryonic axes during seed development, but appeared in cotyledons only at the early stage of development and became undetectable in the cotyledons during late embryogenesis. The expression of the NnMSD1 gene in germinating embryonic axes, in response to various stresses such as heat shock, chilling, and exposure to stress-related chemicals, was also studied. Heat shock strongly inhibited the expression of the NnMSD1 gene, whereas the NnMSD1 transcript level increased strongly in chilling stress treatment. An increase in expression was also highly induced by H2O2 in germinating embryonic axes. The results suggest that the expression pattern of the NnMSD1 gene differed between developing axes and cotyledons, and that the NnMSD1 gene expression responds strongly to chilling and oxidative stress.

Up-regulated manganese superoxide dismutase expression increases apoptosis resistance in human esophageal squamous cell carcinomas

Background Esophageal cancer is one of the most common malignancies in the world. In order to identify the proteins associated with esophageal squamous cell carcinomas （ESCC）, we analyzed the protein profiles of ESCC cases with tumor and matched adjacent normal tissues. Methods Two-dimensional electrophoresis （2-DE） was carried out to analyze the protein profiles. Dysregulated protein spots were identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight （MALDI-TOF） and verified by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry （LC-ESI-IT MS）. RT-PCR and immunohistochemistry on tissue microarray were performed to confirm the gene dysregulation in esophageal cancerous tissues. RNA interference （RNAi） was used to knock down the gene expression in ESCC cell lines. Apoptosis assay with annexin V-FITC/PI staining was conducted and cells were analyzed by flow cytometry. Results 2-DE showed that two protein spots with approximate molecular weights and different pl were elevated in 12 out of 18 ESCCs as compared to the corresponding normal tissues. Both the two spots were identified as MnSOD by MALDI-TOF and were verified by LC-ESI-IT MS. MnSOD overexpression was detected in 14 tumors out of 24 cases by RT-PCR and 52 tumors out of 116 cases by immunohistochemistry comparing to normal epithelia, siRNA-mediated silencing of MnSOD in KYSE450 and KYSE150 cell lines revealed that MnSOD protected ESCC cells from apoptosis induced by ultraviolet （UV） and doxorubicin （DOX）. Conclusions These findings suggest that there existed two isoforms of MnSOD protein in normal and tumor esophageal tissues. MnSOD was overexpressed in ESCC and its up-regulation in esophageal cancer cells was associated with apoptosis resistance.

Mn-SOD and CuZn-SOD polymorphisms and interactions with risk factors in gastric cancer

AIM: To investigate the effects of superoxide dismutase (SOD) polymorphisms (rs4998557 , rs4880), Helicobacter pylori (H. pylori ) infection and environmental factors in gastric cancer (GC) and malignant potential of gastric precancerous lesions (GPL). METHODS: Copper-zinc superoxide dismutase (SOD1, CuZn-SOD)-G7958A (rs4998557 ) and manganese superoxide dismutase (SOD2, Mn-SOD)-Val16Ala (rs4880 ) polymorphisms were genotyped by SNaPshot multiplex polymerase chain reaction (PCR) in 145 patients with GPL (87 cases of gastric ulcer, 33 cases of gastric polyps and 25 cases of atrophic gastritis), 140 patients with GC and 147 healthy controls. H. pylori infection was detected by immunoblotting analysis. RESULTS: The SOD1-7958A allele was associated with a higher risk of gastric cancer [odds ratio (OR) = 3.01, 95% confidence intervals (95% CI): 1.83-4.95]. SOD216Ala/Val genotype was a risk factor for malignant potential of GPL (OR = 2.04, 95% CI: 1.19-3.49). SOD216Ala/genotype increased the risk of gastric cancer (OR = 2.85, 95% CI: 1.66-4.89). SOD1-7958A/genotype, SOD2-16Ala/genotype, alcohol drinking, positive family history and type Ⅰ H. pylori infection were associated with risk of gastric cancer, and there were additive interactions between the two genotypes and the other three risk factors. SOD2-16Ala/Val genotype and positive family history were associated with malignant potential of GPL and jointly contributed to a higher risk for malignant potential of GPL (OR = 7.71, 95% CI: 2.10-28.22). SOD1-7958A/genotype and SOD2-16Ala/genotype jointly contributed to a higher risk for gastric cancer (OR = 6.43, 95% CI: 3.20-12.91). CONCLUSION: SOD1-7958A/and SOD2-16Ala/-genotypes increase the risk of gastric cancer in Chinese Han population. SOD2-16Ala/-genotype is associated with malignant potential of GPL.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats

Objective The roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting （HU） rats. However, the underlying mechanism remains to be established. Methods We investigated the generation of vascular reactive oxygen species （ROS）, Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase （MnSOD） and glutathione peroxidase-1 （GPx-1） mRNA levels in cerebral and mesenteric smooth muscle cells （VSMCs） of HU rats. Results ROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats. Conclusion These results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.