Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle.
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ABSTRACT:
SUBMITTER: Duff DA
PROVIDER: S-EPMC1158560 | biostudies-other | 1982 Jul
REPOSITORIES: biostudies-other
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Project description:A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.
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Project description:Tonsillectomy is a frequently used, low-risk surgical procedure. The post-tonsillectomy haemorrhage occurs rarely, but is a life-threatening complication. Some studies show that the surgical technique affects the haemorrhage rate.To analyse the post-tonsillectomy haemorrhage rate, and to determine whether the effect of the surgical technique on the haemorrhage rate exists.We retrospectively reviewed data of all patients who underwent a tonsillectomy in three regional ENT departments in Bosnia and Herzegovina (Tuzla, Zenica and Bihac) between January 1st 2015 and October 31st 2016. Disorders which could affect the post-tonsillectomy haemorrhage rate were excluded. Tonsillectomy techniques used in these three centers were the hot technique (monopolar/bipolar forceps dissection and haemostasis) and the combined technique (cold steel dissection with monopolar/bipolar forceps haemostasis).1087 patients that underwent a tonsillectomy were analysed in this study. 864 (79.48%) of those were children. 922 (84.82%) patients were operated using the combined technique, 165 (15.17%) underwent a tonsillectomy using the hot technique. Post-tonsillectomy haemorrhage occured in 46 (4.23%) patients. 45 (4.88%) patients had a postoperative haemorrhage after tonsillectomy using the combined technique, whereas haemorrhage occured in 1 patient (0.6%) after using the hot technique. The haemorrhage rate was about eight times lower after tonsillectomy using the hot technique (p=0.012).We conclude that the surgical technique used for tonsillectomy and adenotonsillectomy with the lowest post-tonsillectomy haemorrhage rate is the hot technique; these results are statistically significant. This technique should be used whenever possible, in order to lower the risk of post-tonsillectomy haemorrhage.
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Project description:The success of Agrobacterium mediated plant transformation depends to a certain extent on appropriate selection of the A. tumefaciens strain for a particular plant species. Many stages in a plant transformation procedure are prone to bacterial contamination with similar antibiotic resistance that may compromise the identity of the A. tumefaciens strain used, in turn adversely affecting success of a transformation experiment. Different primer sets were designed to exploit genetic differences among different strains of A. tumefaciens which are commonly used for plant genetic transformation, to identity confirmation as well as to distinguish them from one another. The primer sets Ach5FtsZ-F/R specific for Ach5 and C58GlyA-F/R specific for C58 were designed on chromosomal DNA while primer sets pTiBo542-F/R and nptI-F/R specific for plasmid pTiBo542 are capable to identify and distinguish these strains from one another. These primer sets when used simultaneously in multiplex PCR, produce a pattern which uniquely identifies all these strains and distinguishes them except for GV3101 and C58C1, which can further be distinguished from each other by rifampicin screening. The multiplex PCR assay and primers being reported here serve as a valuable tool in determining the identity of A. tumefaciens strains at any stage of plant transformation procedure.
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Project description:A green extraction process using only water was proposed for the simultaneous extraction of alginate and bioactive compounds from Laminaria ochroleuca. Operation was carried out during non-isothermal heating up to maximal temperatures over the range of 70 °C to 100 °C. Once separated, the alginate and the crude extract were characterised and the biological activities and cytotoxicity of the extracts was studied, the latter in intestinal epithelial cells. Those alginates obtained at 90 °C exhibited the highest extraction yields and viscoelastic features of the corresponding hydrogels. The obtained results show that the extracts obtained by non-isothermal extraction were suitable to formulate nanoparticles, which showed the smallest size (≈ 250-350 nm) when the higher content of fucoidan extract was present. Given the evidenced properties, the extracts may find an application in the formulation of nanoparticulate carriers for drug delivery.
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Project description:Currently, cancer volume in prostate biopsy samples is commonly calculated as linear length of carcinoma divided by total core length and reported as percentage involvement. The measurement of the linear length of carcinoma can be problematic particularly when there are two or more separate foci of carcinoma in a single core. There are two most methods commonly used by practicing pathologists. One method is to measure the exact linear extent of each discrete carcinoma foci in millimeters and then add up the linear length (the exact method, E method). The other method is to measure the core length encompassing all carcinoma foci including the intervening benign prostate tissue (glands and/or stroma) (the scattered method, S method). In this study, we used digital pathology to compare the site-specific and overall cancer volumes measured with the E and S methods and analyzed their correlation with the cancer volume in the corresponding prostatectomy specimens. Our results showed that prostate-cancer volumes estimated with both E and S methods on biopsy samples positively correlate with cancer volume at radical prostatectomy. However, the cancer volumes measured with both E and S methods in the majority of biopsy samples were significantly larger than that in prostatectomy (P<0.001). The E method more closely predicts the cancer volume compared to the S method. The overall cancer volume is better than site-specific cancer volume at biopsy in predicting cancer volume at prostatectomy.
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Project description:The harmonized procedures in terms of the sampling, sample treatment and identification of microplastics in different environmental samples are missing, which poses challenges to researchers to compare the results or to adopt 'the most effective' monitoring approach. Furthermore, in the related literature, the used procedures are rarely tested with spiked microplastics to predetermine their recovery rates. Without this knowledge, results should only be discussed as rough estimations of the real environmental concentrations of microplastics. In this study, six different methods previously used in microplastic studies of different media were tested with municipal wastewater and digested sludge samples, spiked with seven different types of plastic particles and fibres. Recovery rates, time consumption, advantages and disadvantages were assessed and most suitable treatment procedures (i.e. high recovery rates in short amount of time) were chosen for both wastewater and sludge. Suitability of staining with Rose Bengal was examined together with most efficient methods, but it did not improve the recovery of microplastics. In addition, the possible impacts of the treatments for identification with micro-Raman and FTIR microscope were assessed. Filtration with size fractioning was found to be the best method for both wastewater and sludge samples, with recovery rates of spiked microplastics around 91.4% and 92.9%, respectively.
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Project description:BackgroundThe availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media.ResultsAn LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction.ConclusionsThe biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.
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Project description:In order to develop a simple, reliable and low cost enzymatic method for the determination of phenolic compounds we studied polyphenol oxidase activity of crude eggplant (S. melongena) extract using 13 phenolic compounds. Catechol, caffeic and chlorogenic acids, and L-DOPA have been rapidly oxidized with the formation of colored products. Monophenolic compounds have been oxidized at a much slower speed. Ferulic acid, quercetin, rutin, and dihydroquercetin have been found to inhibit polyphenol oxidase activity of crude eggplant extract. The influence of pH, temperature, crude eggplant extract amount, and 3-methyl-2-benzothiazolinone hydrazone (MBTH) concentration on the oxidation of catechol, caffeic acid, chlorogenic acid, and L-DOPA has been investigated spectrophotometrically. Michaelis constants values decrease by a factor of 2 to 3 in the presence of MBTH. Spectrophotometric (cuvette and microplate variants) and smartphone-assisted procedures for phenolic compounds determination have been proposed. Average saturation values (HSV color model) of the images of the microplate wells have been chosen as the analytical signal for smartphone-assisted procedure. LOD values for catechol, caffeic acid, chlorogenic acid, and L-DOPA equaled 5.1, 6.3, 5.8 and 30.0 µM (cuvette procedure), 12.2, 13.2, 13.2 and 80.4 µM (microplate procedure), and 23.5, 26.4, 20.8 and 120.6 µM (smartphone procedure). All the variants have been successfully applied for fast (4-5 min) and simple TPC determination in plant derived products and L-DOPA determination in model biological fluids. The values found with smartphone procedure are in good agreement with both spectrophotometric procedures values and reference values. Using crude eggplant extract- mediated reactions combined with smartphone camera detection has allowed creating low-cost, reliable and environmentally friendly analytical method for the determination of phenolic compounds.
| S-EPMC6930448 | biostudies-literature