A colorimetric sulfo-phospho-vanillin (SPV) method was used to validate a high throughput method for total lipids analysis in fresh and lyophilized oleaginous microorganisms. This method uses a small amount of biological material, does not require a lot of sample manipulation, can be automated, is reproducible and easy to implement. A 96-well microplate SPV assay allows one to determine quickly total lipids in lyophilized cells of oleaginous yeast and microalgae. The new assay method possesses many advantages compared to the others described in the literature: requires a small amount sample, less time (around 1 h) and less labor and does not require organic reagents like chloroform in the reaction.
Strong catalase activity was secreted by Bacillus subtilis cells during stationary growth phase in rich medium but not in sporulation-inducing medium. N-terminal sequencing indicated that the secreted activity was due to the vegetative catalase KatA, previously considered an endocellular enzyme. Extracellular catalase protected B. subtilis cells from oxidative assault. The molecular response to oxidative stress has been extensively studied as it occurs in both gram-negative (6, 17) and gram-positive (18) bacteria. Exposure of aerobically grown Escherichia coli cells to hydrogen peroxide results in the coordinate expression of nine genes that compose the oxyR global regulon (17). A member of this regulon is katG, the structural gene for hydrogen peroxidase I, an H 2 O 2-inducible catalase. The katE gene, coding for the second catalase, HPII, is expressed when cells enter stationary phase and is under the transcriptional control of a specific sigma factor (s) of RNA polymerase (12). For Bacillus subtilis only a single catalase is found in cell extracts during vegetative growth (KatA). Expression of its structural gene, katA, is H 2 O 2 and growth phase inducible (18). A second catalase is found only after the onset of sporulation (13). Catalases and peroxidases in a number of microorganisms, including Haemophilus influenzae (1, 2), Mycobacterium tuberculosis (10), and Streptomyces viridosporus (16), have been studied. These enzymes play important roles in several microbial processes also independent of oxidative stress. Microbial peroxidases are involved in the oxidation of a large number of aromatic compounds, including recalcitrant substances (14). Catalases of pathogenic bacteria, like Listeria monocytogenes, H. influenzae, and Helicobacter pylori, are considered potential virulence factors which may counteract the oxygen-dependent defense mechanisms of infected phagocytes (1, 8, 15). It is thought that during food fermentation catalases from lactobacilli decrease hydrogen peroxide accumulation, which would severely affect the sensory quality of products (11). Catalases from Aspergillus niger are used to eliminate from milk hydrogen peroxide either endogenously produced or added in the process of cold pasteurization. We have found a vegetative catalase activity secreted by B. subtilis cells during the stationary growth phase. We report here that this activity is due to KatA and that its secretion is essential to ensure complete protection of B. subtilis cells against oxidative stress. The finding that KatA is a secreted enzyme will facilitate its purification and the study of its mechanism of action, therefore enhancing interest in B. subtilis catalase. KatA, the vegetative catalase of B. subtilis, is secreted during the stationary growth phase. Filter-sterilized cell-free super
-With the objective to promote in vitro callus induction, cotyledon and hypocotyl segments of "perennial soybean" (Glycine wightii (Wight & Arn.) Verdc.) were inoculated in basal medium MS supplemented with sucrose (1.5 e 3%) and 0.8% agar and different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6 -furfurylaminopurine (kinetin). The explants were maintained in a dark growth room at 28 o C. The best callus induction was observed in explants (cotyledon and hypocotyl) maintained in medium containing the combination of 2,4-D (1 mg.L -1 ), kinetin (0.1 mg.L -1 ) and 3% sucrose. To promote callus subculture, the MS
Cotyledon explants were first cultured on MS medium supplemented with 4.52 M 2,4-D and 0.46 mM kinetin. The development of the calli was followed (0, 4, 8, 12, 16, 20, 24, 28 and 32 days after) and the growth curve was determined, based in fresh and dry weight. The growth curve presented sigmoidal form with four distinct phases. The highest growth percentage was observed at the exponential phase and the lowest at the stationary phase. These results indicated that cotyledon callus subculture should be performed 20 days after inoculation. The calli obtained after a period of 28 days were freeze dried, macerated and submitted to extraction with buffers of different pH values (2.6; 4.0; 6.0; 8.0 and 10.0) and the proteins in the extracts were determined by Bradford method. The pH 8.0 buffer was the most efficient to extract the largest amount of protein. The amino acid analyses calli showed a high content of aspartic acid and low content of metionin. The dynamics of protein synthesis in calli was followed by SDS-PAGE electrophoresis.Index terms: Glycine, Leguminosae, callogenesis, SDS-PAGE. RESUMOExplantes cotiledonários foram primeiramente cultivados em meio MS suplementado com 2,4-D 4,52 mM e cinetina 0,46 mM. O desenvolvimento dos calos foi acompanhado (0, 4, 8, 12, 16, 20, 24, 28 até 32 dias após o subcultivo) e uma curva de crescimento foi determinada com base nos pesos fresco e seco dos calos. A curva de crescimento apresentou um padrão sigmoidal com quatro fases distintas. A maior porcentagem de crescimento foi observada na fase exponencial e a menor na fase estacionária. Estes resultados indicam que o subcultivo dos calos pode ser realizado 20 dias após a inoculação. Os calos obtidos após um período de 28 dias foram liofilizados, macerados e submetidos à extração de proteínas solúveis, utilizando tampões com diferentes valores de pH (2,6; 4,0; 6,0; 8,0 e 10,0) e o teor protéico dos extratos foi determinado pelo método de Bradford. O tampão pH 8,0 foi o mais eficiente na extração de proteínas solúveis dos calos. A análise de aminoácidos mostrou que os calos apresentam um alto teor de ácido aspártico/ asparaina em um baixo teor de metionina. A dinâmica da síntese de proteínas nos calos foi acompanhada por ensaios eletroforéticos (SDS-PAGE).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.