scholarly journals Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii

2003 ◽  
Vol 369 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Christina SCHLÜPEN ◽  
Maria A. SANTOS ◽  
Ulrike WEBER ◽  
Albert de GRAAF ◽  
José L. REVUELTA ◽  
...  
Keyword(s):  
Carbon Flux ◽  
Specific Activity ◽  
Serine Hydroxymethyltransferase ◽  
Detectable Effect ◽  
Ashbya Gossypii ◽  
Gene Encoding ◽  
13C Labelling ◽  
Glycine Metabolism ◽  
Aldolase Activity

Riboflavin overproduction in the ascomycete Ashbya gossypii is limited by glycine, a precursor of purine biosynthesis, and therefore an indicator of glycine metabolism. Disruption of the SHM2 gene, encoding a serine hydroxymethyltransferase, resulted in a significant increase in riboflavin productivity. Determination of the enzyme's specific activity revealed a reduction from 3m-units/mg of protein to 0.5m-unit/mg protein. The remaining activity was due to an isoenzyme encoded by SHM1, which is probably mitochondrial. A hypothesis proposed to account for the enhanced riboflavin overproduction of SHM2-disrupted mutants was that the flux from glycine to serine was reduced, thus leading to an elevated supply with the riboflavin precursor glycine. Evidence for the correctness of that hypothesis was obtained from 13C-labelling experiments. When 500μM 99% [1-13C]threonine was fed, more than 50% of the label was detected in C-1 of glycine resulting from threonine aldolase activity. More than 30% labelling determined in C-1 of serine can be explained by serine synthesis via serine hydroxymethyltransferase. Knockout of SHM1 had no detectable effect on serine labelling, but disruption of SHM2 led to a decrease in serine (2—5%) and an increase in glycine (59—67%) labelling, indicating a changed carbon flux.

An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

1983 ◽  
Vol 50 (03) ◽  
pp. 740-744 ◽  
Author(s):  
Nils Bergsdorf ◽  
Torbjörn Nilsson ◽  
Per Wallén
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Thromboembolic Disease ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture

2020 ◽  
Vol 36 (3) ◽  
pp. 82-89
Author(s):  
O.V. Gromova ◽  
O.S. Durakova ◽  
S.V. Generalov ◽  
L.F. Livanova ◽  
O.A. Volokh
Keyword(s):  
Cell Culture ◽  
Vibrio Cholerae ◽  
Production Control ◽  
Specific Activity ◽  
Methodological Approach ◽  
Toxin Production ◽  
Submerged Cultivation ◽  
Vaccine Production ◽  
Antigenic Activity

Том 36(2020) №3 стр. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89А.В. Гаева1*, О.В. Громова1, О.С. Дуракова1, С.В. Генералов1, Л.Ф. Ливанова1, О.А. Волох1 Определение специфической активности компонентов холерной химической вакцины с использованием культуры клеток 1ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб»» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, Саратов 410005 *[email protected] Поступила - 2019-11-26; После доработки - 2020-03-16; Принята к публикации - 2020-05-15 Список литературы Описаны методы определения динамики продукции токсинов штаммом Vibrio cholerae 569B при глубинном культивировании в биореакторе и антигенной активности специфической фракции холерогена-анатоксина по анатоксинсвязыванию с использованием клеточных культур. Показана высокая степень соответствия результатов, полученных методами, применяемыми для контроля этапов производства холерной химической вакцины и рассмотренными в данной работе. Отмечено, что применение клеточной линии СНО-К1 наиболее перспективно для замены биомоделей на промежуточных этапах контроля активных компонентов холерной химической вакцины. Разработанный методический подход впервые предлагается использовать на этапах производства холерной бивалентной химической вакцины. культура клеток, Vibrio cholerae, холерная химическая вакцина, контроль производства, холера. Vol 36(2020) N 3 p. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89A.V. Gaeva1*, O.V. Gromova1, O.S. Durakova1, S.V. Generalov1, L.F. Livanova1, O.A. Volokh1 Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture 1Russian Research Anti-Plague Institute «Microbe» of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Saratov, 410005 *[email protected] Received - 26.11.2019; Accepted - 15.05.2020 References The methods has been described to determine the dynamics of toxin production by the Vibrio cholerae 569B strain during submerged cultivation in bioreactor and of the antigenic activity of specific choleragen anatoxin fraction by anatoxin binding levels using cell cultures. High degree of consistency was observed between the results obtained via the method under consideration and those obtained via control methods at different stages of cholera chemical vaccine production. It was shown that the CHO-K1 cell line is the most promising substitute for biomodels at the intermediate stages of control of active cholera chemical vaccine components. The developed methodological approach was first proposed for use at the stages of cholera chemical bivalent vaccine manufacturing. cell culture, Vibrio cholerae, cholera chemical vaccine, production control, cholera.

The Natural Radioactivity in Food: A Comparison Between Different Feeding Regimes

2019 ◽  
Vol 15 (5) ◽  
pp. 493-499 ◽  
Author(s):  
Francesco Caridi ◽  
Santina Marguccio ◽  
Alberto Belvedere ◽  
Maurizio D`Agostino ◽  
Giovanna Belmusto
Keyword(s):  
Vegetable Oils ◽  
Natural Radioactivity ◽  
Specific Activity ◽  
Mean Value ◽  
Vegetable Food ◽  
Fish Samples ◽  
Feeding Regimes ◽  
Italian Origin ◽  
Dose Levels

Background: In this article a comprehensive study was carried out for the determination of natural radioactivity in animal and vegetable food (meat, fish, milk and derivates, legumes, cereals and derivates, fruit, hortalizas, vegetables, vegetable oils) typical of different feeding regimes, for the age category higher than 17 years. Methods: A total of eighty-five samples of Italian origin, coming from large retailers during the years 2014, 2015 and 2016, were analyzed through HPGe gamma spectrometry. Results: The specific activity of 40K was investigated and its mean value was found to be: (106.3 ± 6.9) Bq/kg for bovine, swine and sheep meat; (116.5 ± 9.7) Bq/kg for fish; (52.9 ± 3.1) Bq/kg for milk and derivates; (271.9 ± 16.7) Bq/kg for legumes; (67.2 ± 4.7) Bq/kg for cereals and derivates; (52.7 ± 4.4) Bq/kg for fruit; (72.9 ± 5.6) Bq/kg for hortalizas; (83.9 ± 6.5) Bq/kg for vegetables; lower than the minimum detectable activity for vegetable oils. For animal food the highest mean 40K activity concentration was found in fish samples; for vegetable food the highest one was detected in legumes. Conclusion: The evaluation of dose levels due to the food ingestion typical of Mediterranean, Vegetarian and Vegan diets was performed. The annual effective dose was found to be 0.16 mSv/y, 0.41 mSv/y and 0.54 mSv/y, respectively.

Inactivation or amplification of the spa2 gene, encoding a potential stationary-phase regulator, affects differentiation in Streptomyces ambofaciens

1997 ◽  
Vol 43 (12) ◽  
pp. 1118-1125 ◽  
Author(s):  
Martine Aubert ◽  
Elisabeth Weber ◽  
Brigitte Gintz ◽  
Bernard Decaris ◽  
Keith F. Chater
Keyword(s):  
Stationary Phase ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Detectable Effect ◽  
Multicopy Plasmid ◽  
Gene Encoding ◽  
Wild Type Phenotype ◽  
Streptomyces Ambofaciens ◽  
Mycelial Development

The deduced product of the spa2 gene of Streptomyces ambofaciens is a homologue of RspA, involved in stationary-phase σs factor regulation in Escherichia coli. This suggests that Spa2 could play a part in stationary-phase-associated differentiation in S. ambofaciens. The disruption of spa2 led to reductions in aerial mycelial development and associated spore pigmentation. The mutant phenotype reverted to the wild-type phenotype when the disruption construct spontaneously excised. The spa2 disruption had no detectable effect on growth rates in different media or antibiotic production and resistance. When spa2 was placed on a multicopy plasmid, a severe defect in formation and pigmentation of aerial mycelium resulted. These results strongly suggest that Spa2 is involved in a complex manner in the morphological differentiation process.Key words: Streptomyces, differentiation, stationary-phase regulator.

The determination of the specific activity of plasma glycerol

1982 ◽  
Vol 60 (6) ◽  
pp. 856-858 ◽  
Author(s):  
Clément Gauthier ◽  
Ross Layberry
Keyword(s):  
Column Chromatography ◽  
Turnover Rate ◽  
Specific Activity ◽  
Anionic Exchange ◽  
Removal Of Contaminants ◽  
The One ◽  
Exchange Resins

A method for the determination of the specific activity of plasma glycerol is described. Anionic contaminants are first removed from deproteinized plasma by anionic exchange resins (treated plasma). Glycerol in treated plasma is then quantitatively converted to glycerol-3-phosphate (G3P), which is isolated by column chromatography and counted for 14C radioactivity. The specific activity thus calculated was 100.1 ± 2.9% of a standard of known specific activity. When the specific-activity of glycerol is determined from plasma without prior removal of anionic contaminants (untreated plasma), the calculated specific activity is 1.99 ± 0.15 times higher than the one calculated after their removal. Omission of the removal of contaminants leads to a near 100% error in the calculation of the turnover rate of glycerol.not available

On the Determination of Human Saliva Aldolase Activity

1957 ◽  
Vol 36 (6) ◽  
pp. 852-861 ◽  
Author(s):  
Kirk C. Hoerman ◽  
William E. Robinson
Keyword(s):  
Human Saliva ◽  
Aldolase Activity

Determination of the specific ?-activity of Pu239 and Pu240

Atomic Energy ◽  
1960 ◽  
Vol 6 (1) ◽  
pp. 41-42
Author(s):  
Ya. P. Dokuchaev
Keyword(s):  
Specific Activity

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

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