Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay

Kuo, Wen-Yu; Huang, Chien-Hua; Shih, C H.; Jinn, Tsung-Luo

doi:10.21769/bioprotoc.811

Cited by 17 publications

(16 citation statements)

References 1 publication

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“…After separation, the gel was stained for SOD activity essentially as described in ref. 96 . FeSOD enzymes are inhibited by peroxide but not by KCN, which however inhibits MnSOD enzymes.…”

Section: Resultsmentioning

confidence: 99%

Extensive gain and loss of photosystem I subunits in chromerid algae, photosynthetic relatives of apicomplexans

Sobotka

Esson

Koník

et al. 2017

Sci Rep

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In oxygenic photosynthesis the initial photochemical processes are carried out by photosystem I (PSI) and II (PSII). Although subunit composition varies between cyanobacterial and plastid photosystems, the core structures of PSI and PSII are conserved throughout photosynthetic eukaryotes. So far, the photosynthetic complexes have been characterised in only a small number of organisms. We performed in silico and biochemical studies to explore the organization and evolution of the photosynthetic apparatus in the chromerids Chromera velia and Vitrella brassicaformis, autotrophic relatives of apicomplexans. We catalogued the presence and location of genes coding for conserved subunits of the photosystems as well as cytochrome b6f and ATP synthase in chromerids and other phototrophs and performed a phylogenetic analysis. We then characterised the photosynthetic complexes of Chromera and Vitrella using 2D gels combined with mass-spectrometry and further analysed the purified Chromera PSI. Our data suggest that the photosynthetic apparatus of chromerids underwent unique structural changes. Both photosystems (as well as cytochrome b6f and ATP synthase) lost several canonical subunits, while PSI gained one superoxide dismutase (Vitrella) or two superoxide dismutases and several unknown proteins (Chromera) as new regular subunits. We discuss these results in light of the extraordinarily efficient photosynthetic processes described in Chromera.

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“…After separation, the gel was stained for SOD activity essentially as described in ref. 96 . FeSOD enzymes are inhibited by peroxide but not by KCN, which however inhibits MnSOD enzymes.…”

Section: Resultsmentioning

confidence: 99%

Extensive gain and loss of photosystem I subunits in chromerid algae, photosynthetic relatives of apicomplexans

Sobotka

Esson

Koník

et al. 2017

Sci Rep

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“…S OD activity was photochemically assayed on 10% polyacrylamide gels (NATIVA-PAGE) based on the nitro blue tetrazolium (NBT) photoreaction and in Tris-glycine buffer under non-denaturing conditions according (Kuo et al .,. 2013; Weydert and Cullen 2010).…”

Section: Methodsmentioning

confidence: 99%

Adaptive stress response induced by toluene increasesSporothrix schenckiivirulence and host immune response

Téllez-Martínez

Batista-Duharte

Silva

et al. 2019

Preprint

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24Environmental factors modify the physiology of microorganisms, allowing their survival in 25 extreme conditions. However, the influence of chemical contaminants on fungal virulence has 26 been little studied. Sporotrichosis is an emergent fungal disease caused by Sporothrix schenckii, 27 a soil-inhabiting fungus that has been found in polluted environments. Here, we evaluated the 28 adaptive stress response of S. schenckii induced by toluene, a key soil contaminant. The effect 29 on fungal virulence and host immune response was also assessed. The fungus survived up to 30 0.10% toluene in liquid medium. Greater production of melanosomes and enhanced activity 31 superoxide dismutase, associated to increased tolerance to H2O2 were observed in toluene-32 exposed fungi. Intraperitoneal infection of mice with S. schenckii treated with either 0, 0.01 or 33 0.1% of toluene, resulted in greater fungal burden at day 7 post-infection in spleen and liver in 34 the groups infected with fungus treated with toluene 0.1%. A higher production of Il-1β, TNF-35 α, IL-10 and nitric oxyde by peritoneal macrophages and IFNɣ, IL-4 and IL-17 by splenocytes 36 was also observed in that group. Our findings showed that morphological and functional 37 changes induced by toluene leads to increased S. schenckii virulence and antifungal host 38 immune response in our model. 39 40 41 42 43 44 45 78 Aromatic compounds such as benzene, toluene, ethylbenzene and xylene isomers, 79 collectively known as BTEX, are one of the major contributors to environmental pollution. 80 BTEX are extensively used as solvents in many industrial processes and as base reagents for 81 the production of chemicals products. In addition, BTEX are components of gasoline and 82 aviation fuels. They are often released in the environment during production, transport, use and 83 disposal, causing groundwater, surface water, and soil contamination. Toluene has been found 84 in at least 1,012 of the 1,699 National Priorities List sites identified by the Environmental 85 Protection Agency (EPA) and it is included in the Agency for Toxic Substances and Disease 86 Registry (ATSDR) 2017 substance priority list, which is based on a combination of their 87 frequency, toxicity, and potential for human exposure (Agency for Toxic Substances and 88 Disease Registry. ATSDR's Substance Priority List. 2017).89Prenafreta-Boldu isolated several fungi, including S. schenckii, in biofilters exposed to 90 polluted air or hydrocarbon gas streams. The data from this research showed that volatile 91 hydrocarbon-degrading strains are closely related to a very restricted number of pathogenic 92 fungal species that cause severe mycoses, especially neurological infections, in 93 immunocompetent individuals (Prenafeta-Boldú et al., 2006). 94To date, there have been no studies examining the influence of chemical contaminants 95 on the biology of S. schenckii sensu latu. Given that this fungus inhabits soils and this niche is 96 frequently contaminated with oil and their products, the objective of this...

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“…Protein extracts were prepared in 25 mmol/L HEPES pH 7.8, 2% PVPP, 0.2 mmol/L EDTA and quantified by Bradford assay. In-gel superoxide dismutase (SOD) activity assays were performed according to (Kuo et al 2013) with minor modifications. About 20 lg protein extracts, supplemented with SDS-free protein loading dye, were separated on 10% native polyacrylamide gels at 4°C, 12 mA for 2 h. The gels had been prerun for 2 h to remove TEMED and APS.…”

Section: Superoxide Dismutasementioning

confidence: 99%

Antioxidative responses during germination in quinoa grown in vitamin B‐rich medium

Pitzschke

Fraundorfer²,

Guggemos³

et al. 2015

Food Science & Nutrition

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Synthetic vitamin preparations have grown in popularity to combat health risks associated with an imbalanced diet, poor exercise and stress. In terms of bioavailability and diversity, they lack behind vitamins naturally occurring in plants. Solutions to obtain plant-derived vitamins at a larger scale are highly desirable. B vitamins act as precursors of enzymatic cofactors, thereby regulating important metabolic processes both in animals and plants. Because during plant germination, the vitamin content and micronutrient availability increase, sprouts are generally considered a healthier food as compared to dry grains. Germination only occurs if a plant′s antioxidant machinery is sufficiently activated to cope with oxidative stress. Seeds of quinoa, an edible gluten-free plant naturally rich in minerals, germinate readily in a solution containing the eight B vitamins. We studied biochemical changes during quinoa germination, with a focus on nutritionally relevant characteristics. The results are considered from a nutritional and plant physiological perspective. Germination of quinoa in vitamin-rich medium is a promising strategy to enhance the nutritional value of this matrix. Additional health-beneficial effects indirectly resulting from the vitamin treatment include elevated levels of the multi-functional amino acid proline and a higher antioxidant capacity. Plant biomolecules can be better protected from oxidative damage in vivo.

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Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay

Cited by 17 publications

References 1 publication

Extensive gain and loss of photosystem I subunits in chromerid algae, photosynthetic relatives of apicomplexans

Extensive gain and loss of photosystem I subunits in chromerid algae, photosynthetic relatives of apicomplexans

Adaptive stress response induced by toluene increasesSporothrix schenckiivirulence and host immune response

Antioxidative responses during germination in quinoa grown in vitamin B‐rich medium

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