scholarly journals Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes

2000 ◽  
Vol 350 (2) ◽  
pp. 429-441 ◽  
Author(s):  
Stuart M. PITSON ◽  
Richard J. D'ANDREA ◽  
Lucianne VANDELEUR ◽  
Paul A. B. MORETTI ◽  
Pu XIA ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mammalian Cells ◽  
Active Form ◽  
Chemical Properties ◽  
Sphingosine Kinase ◽  
Exchange Chromatography ◽  
Recombinant Enzymes ◽  
Post Translational Modifications ◽  
E Coli ◽  
Physico Chemical

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 106-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

Ultralight ultrafast enzymes

2021 ◽  
Author(s):  
Xuepei Zhang ◽  
Zhaowei Meng ◽  
Christian Beusch ◽  
Hassan Gharibi ◽  
Qing Cheng ◽  
...  
Keyword(s):  
Nitrogen Isotopes ◽  
Chemical Properties ◽  
Reaction Rates ◽  
Inorganic Materials ◽  
Recombinant Enzymes ◽  
E Coli ◽  
Physico Chemical ◽  
Structural Entropy ◽  
Heavy Carbon ◽  
Normal Enzyme

Abstract Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physico-chemical properties from their isotopically natural (native) counterparts; however, in biotechnology such effects have not been investigated yet. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in ultralight media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase showed a 40-250% increase in activity compared to the native counterparts. The efficiency of ultralight Pfu DNA polymerase in polymerase chain reaction was also significantly higher than that of the normal enzyme. At 10 °C, the advantage factor of ultralight enzymes typically increased by 50%, which points towards the reduction in structural entropy as the main factor explaining the kinetic effect of heavy isotope depletion. Ultralight enzymes may find an application where extreme reaction rates are required.

scholarly journals Chikungunya E2 Protein Produced in E. coli and HEK293-T Cells—Comparison of Their Performances in ELISA

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 939
Author(s):  
Flávia Fonseca Bagno ◽  
Lara Carvalho Godói ◽  
Maria Marta Figueiredo ◽  
Sarah Aparecida Rodrigues Sérgio ◽  
Thaís de Fátima Silva Moraes ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Indirect Elisa ◽  
Acute Onset ◽  
Diagnostic Tools ◽  
Igg Antibodies ◽  
Post Translational Modifications ◽  
E Coli ◽  
Glycosylation Sites ◽  
Igm Elisa ◽  
Antigen Antibody

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)—a prokaryotic system unable to produce post-translational modifications—or in HEK-293T mammalian cells—a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

Reaction of Elemental Sulfur and Mercaptobenzothiazole

1956 ◽  
Vol 29 (4) ◽  
pp. 1369-1372
Author(s):  
G. A. Blokh ◽  
E. A. Golubkova ◽  
G. P. Miklukhin
Keyword(s):  
Elemental Sulfur ◽  
Active Form ◽  
Chemical Properties ◽  
Intermediate Compound ◽  
Point Of View ◽  
Experimental Fact ◽  
Acceleration Process ◽  
Physico Chemical ◽  
Lower Temperature ◽  
Vulcanization Process

Abstract One of the most important problems in the field of the physics and chemistry of rubber is that of vulcanization. Until now no single theory has been established, which elucidates the complex physico-chemical changes which occur during this process. Still more obscure has been the mechanism of the action of vulcanization accelerators, which, as is well known, not only reduce the time and the temperature of vulcanization, but also influence the physico-mechanical and chemical properties of the rubber. Most investigators have assumed that in the acceleration process a reaction with sulfur converts it to an active form which is capable of bringing about vulcanization at a lower temperature and at a greater rate, than with ordinary elemental sulfur in the absence of an accelerator. This point of view is based on the experimental fact that the vulcanization of rubber by sulfur dioxide and hydrogen sulfide, for example, which form sulfur in the nascent condition, proceeds rapidly even at room temperature. Investigators have also assumed that in the vulcanization process activation of sulfur in the presence of accelerators may occur by different mechanisms. It is possible that the accelerator, reacting with elemental sulfur, forms unstable intermediate compounds, which decompose with liberation of sulfur in an active form. The latter reacts with rubber, and the regenerated accelerator reacts again with elemental sulfur, etc. However, a different process is possible for the activation of elemental sulfur. By this second mechanism the unstable combination of accelerator and sulfur reacts directly with rubber without the formation of active sulfur. Both these mechanisms necessarily assume the formation of intermediate unstable combinations of the accelerator with sulfur. However, direct, experimentally-based demonstrations of such an interaction are lacking in the literature. There exist only theoretical hypotheses concerning the nature of the possible intermediate combination of the accelerator with sulfur. According to Ostromislensky's concepts, further developed by Bedford, such an intermediate compound has the character of a polysulfide. According to Bruni and Romani, this intermediate compound is a disulfide. As is well known, the disulfide theory was placed in doubt by Zaide and Petrov on the basis of data from the vulcanization of rubber in the presence of benzothiazolyl disulfide.

scholarly journals Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications

2000 ◽  
Vol 84 (S1) ◽  
pp. 19-25 ◽  
Author(s):  
Klaas D. Kussendrager ◽  
A. C. M. van Hooijdonk
Keyword(s):  
Mammalian Cells ◽  
Bovine Milk ◽  
Chemical Properties ◽  
Mechanisms Of Action ◽  
Industrial Processes ◽  
Physico Chemical ◽  
Lactoperoxidase System ◽  
Wide Range ◽  
Occurrence Mechanism ◽  
Micro Organisms

Lactoperoxidase (LP) is one of the most prominent enzymes in bovine milk and catalyses the inactivation of a wide range of micro-organisms in the lactoperoxidase system (LP-s). LP-systems are also identified as natural antimicrobial systems in human secretions such as saliva, tear-fluid and milk and are found to be harmless to mammalian cells. The detailed molecular structure of LP is identified and the major products generated by the LP-s and their antimicrobial action have been elucidated for the greater part. In this paper several aspects of bovine LP and LP-s are discussed, including physico-chemical properties, occurrence in milk and colostrum and mechanisms of action. Since the introduction of industrial processes for the isolation of LP from milk and whey the interest in this enzyme has increased considerably and attention will be paid to potential and actual applications of LP-systems as biopreservatives in food and other products.

scholarly journals Development of the technology for preparation of enzymatic hydrolysate of waste chick embryos

2021 ◽  
Vol 21 (3) ◽  
pp. 200-205
Author(s):  
Yu. S. Ovsyannikov ◽  
M. S. Dursenev
Keyword(s):  
Nutrient Medium ◽  
Mass Fraction ◽  
Chemical Properties ◽  
Chick Embryos ◽  
Nutrient Media ◽  
Enzymatic Hydrolysate ◽  
E Coli ◽  
Physico Chemical ◽  
Growth Properties ◽  
Solid Nutrient Medium

The development of technologies for preparation of protein nutritional bases for microbiological nutrient media, from production waste of mainly readily available or non-food products, is a promising area in biotechnology. Researchers of Vyatka State Agrotechnological University assume that non-food secondary raw materials, such as waste chick embryos (WCEs) used in the production of anti-influenza products, could be used for these purposes, after removal of the virus-containing allantoic fluid. The aim of the study was to develop a technology for preparation of WCE enzymatic hydrolysate (WCEEH), and to evaluate growth properties of the hydrolysate-based solid nutrient medium, using Escherichia coli M-17 and Pseudomonas alcaligenes IP-1 test strains. Materials and methods: the authors offer methodological approaches to obtaining WCEEH and substantiate hydrolysis parameters. The obtained WCEEH was characterised in terms of physico-chemical properties: pH, amine nitrogen, total nitrogen, sodium chloride, degree of protein cleavage. The growth properties of the hydrolysate-based nutrient medium were studied using E. coli M-17 and Ps. alcaligenes IP-1 test strains. Results: the experiments demonstrated the feasibility of performing enzymatic hydrolysis of WCEs, and assessed physico-chemical properties of the prepared WCEEH batches. The study demonstrated the possibility of using the prepared hydrolysate as a component of solid nutrient media for growing the selected test strains. Conclusions: the study substantiated the optimal technological parameters for WCE enzymatic hydrolysis: pH (7.6 ± 0.3), duration (48 ± 2 h), temperature (49 ± 1) °C. The loading of hydrolysis components was optimised: mass fraction of the substrate—500 g/L, mass fraction of the hydrolysing agent—100 g/L. The physico-chemical properties of WCEEH make it suitable for preparation of microbiological media; the hydrolysate-based solid nutrient medium consistently ensures the growth of E. coli M-17 and Ps. alcaligenes IP-1 test strains with standard properties. The growth properties of the experimental medium are comparable to those of the meat-peptone broth-based nutrient medium.

scholarly journals Applications of In-Cell NMR in Structural Biology and Drug Discovery

2019 ◽  
Vol 20 (1) ◽  
pp. 139 ◽  
Author(s):  
CongBao Kang
Keyword(s):  
Drug Discovery ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Structural Information ◽  
Living Cells ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Nmr Studies ◽  
Post Translational Modifications

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

CHANGES IN MICROBIOLOGICAL AND PHYSICO-CHEMICAL PROPERTIES DURING COMPOSTING OF SIAM WEED (Chromolaena odorata) AND COWDUNG

2018 ◽  
Vol 14 (2) ◽  
pp. 40-48
Author(s):  
A K AKINTOKUN ◽  
P O AKINTOKUN ◽  
A O OBAWUSI ◽  
O R LAWAL
Keyword(s):  
Micrococcus Luteus ◽  
Chemical Properties ◽  
Fungal Species ◽  
Cow Dung ◽  
Chromolaena Odorata ◽  
E Coli ◽  
Standard Procedures ◽  
Physico Chemical ◽  
Three Samples ◽  
Phosphorus And Potassium

Three compost samples were prepared in this study from Siam weed (Chromolaena odorata) and cowdung. Sample A was prepared from Cow dung and siam weed at ratio 100g: 100g, Sample B was prepared from 200g chopped siam weed and sample C contained 200g cowdung. These three sam-ples were composted in plastic drums perforated for aeration and each sample were replicated three times. The content in the drums were regularly turned and monitored at 1, 10, 30 and 60 days for mi-crobiological and physicochemical properties. The microbiological and physicochemical analyses of the compost were carried out using standard procedures. Bacterial, Coliform and Fungal count in-creased from day 1 to the 30th day and thereafter decreased from 30th day to the 60th day in all the composting samples. The bacteria species isolated and identified were Pseudomonas fragilis, Pseu-domonas nitrificans, Proteus mirabilis, E. coli, Streptococcus faecium, Micrococcus luteus, Clostridium perfringes, Bacillus cereus, Proteus morganii, Micrococcus acidophilus. Fungal species were Aspergil-lus flavus, Aspergillus fumigatus, Fusarium oxysporium, Penicillum chrysogenum, Aspergillus niger, Mucor sp. and Saccharomyces cerevisiae. The pH of the composted samples ranges between 5.8 to 6.9. The nitrogen, phosphorus and potassium content increased with days of composting but the heavy metals decreased with days of composting. The sulfatase, phosphatase, dehydrogenase, amyl-ase and cellulose enzymes in the three samples increased from day 1 to the 60th day. Sulfatase en-zyme which was the highest ranged from 25 to 76.5% in the three sample, phosphatase (14 to 60.5%), dehydrogenase (20.5 to 55.0%), cellulose (16.5 to 49%) and amylase which was the least enzyme recorded ranged from 5.0 to 38%.

Physico-chemical properties of actin cleaved with bacterial protease from E. coli A2 strain

FEBS Letters ◽  
1991 ◽  
Vol 279 (1) ◽  
pp. 49-51 ◽  
Author(s):  
S.Yu. Khaitlina ◽  
J.H. Collins ◽  
I.M. Kuznetsova ◽  
V.P. Pershina ◽  
I.G. Synakevich ◽  
...  
Keyword(s):  
Chemical Properties ◽  
E Coli ◽  
Bacterial Protease ◽  
Physico Chemical

Discussion. The fate of antigen

1956 ◽  
Vol 146 (922) ◽  
pp. 34-36 ◽  
Keyword(s):  
Fluorescent Antibody ◽  
Active Form ◽  
Chemical Properties ◽  
Liver Parenchyma ◽  
Reticuloendothelial System ◽  
Immunological Tolerance ◽  
The Body ◽  
Physico Chemical ◽  
Foreign Materials ◽  
Reticulo Endothelial System

I should like to discuss an approach to the problem of immunological tolerance, which is closely akin to Burnet & Fenner’s (1949) ‘self-marker’ hypothesis, although its emphasis is less on the question of how antibodies are produced than on the detailed fate of potentially antigenic molecules when they leave the plasma and lymph and are catabolized. Dr Cinader’s figures certainly suggest that in tolerant rabbits heterologous albumin is eliminated from the plasma at rates which are substantially identical with those of homologous albumin. This is true also of heterologous plasma proteins in normal rabbits during the period before antibodies appear. Furthermore, we have found that in normal rats, in which albumin and globulin are eliminated at markedly different rates (Campbell, Cuthbertson, Matthews & McFarlane 1956), heterologous albumin behaves much like rat albumin and heterologous γ -globulin like rat γ -globulin for a week or more. It is a reasonable working hypothesis that foreign materials in the circulation are in the first place removed from the tissue fluids by the same mechanisms as deal with native materials possessing similar physico-chemical properties (e.g. molecular weight, surface charge). We are largely ignorant of what these mechanisms might be, except for the part played by reticulo-endothelial cells in removing particulate matter and denatured or aggregated proteins. However, the use by Coons, Leduc & Kaplan (1951) of fluorescent antibody to locate antigenic material in histological sections has shown that apparently undenatured proteins (e.g. bovine albumin, human γ -globulin) introduced into the plasma are detectable in immunologically active form within a wide variety of cells, such as renal tubule cells, liver parenchyma or vascular endothelium, in addition to cells of the reticuloendothelial system proper. There are possibly at least two processes going on concurrently: first, a non-selective ingestion of their surrounding fluid (with all that it contains) by cells which are widely distributed in the body, and are not necessarily confined to the reticulo-endothelial system. The mechanism may well be one of vacuolar ingestion (‘pinocytosis’), for which there is some circumstantial evidence, but which also presents difficulties. This would be one mechanism responsible for normal plasma protein catabolism. Secondly, there may be a selective removal of particulate or aggregated material, especially when negatively charged, by cells of the reticulo-endothelial system, notably by liver Küpffer cells.

scholarly journals Impact of Physico-Chemical Properties of Cellulose Nanocrystal/Silver Nanoparticle Hybrid Suspensions on Their Biocidal and Toxicological Effects

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1862
Author(s):  
Dafne Musino ◽  
Julie Devcic ◽  
Cécile Lelong ◽  
Sylvie Luche ◽  
Camille Rivard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Low Cost ◽  
Aqueous Suspension ◽  
Chemical Properties ◽  
Molar Ratio ◽  
Biocidal Activity ◽  
Phagocytic Capacity ◽  
Toxicological Effects ◽  
Physico Chemical

There is a demand for nanoparticles that are environmentally acceptable, but simultaneously efficient and low cost. We prepared silver nanoparticles (AgNPs) grafted on a native bio-based substrate (cellulose nanocrystals, CNCs) with high biocidal activity and no toxicological impact. AgNPs of 10 nm are nucleated on CNCs in aqueous suspension with content from 0.4 to 24.7 wt%. XANES experiments show that varying the NaBH4/AgNO3 molar ratio affects the AgNP oxidation state, while maintaining an fcc structure. AgNPs transition from 10 nm spherical NPs to 300 nm triangular-shaped AgNPrisms induced by H2O2 post-treatment. The 48 h biocidal activity of the hybrid tested on B. Subtilis is intensified with the increase of AgNP content irrespective of the Ag+/Ag0 ratio in AgNPs, while the AgNSphere−AgNPrism transition induces a significant reduction of biocidal activity. A very low minimum inhibitory concentration of 0.016 mg AgNP/mL is determined. A new long-term biocidal activity test (up to 168 h) proved efficiency favorable to the smaller AgNPs. Finally, it is shown that AgNPs have no impact on the phagocytic capacity of mammalian cells.

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