scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nicole L. McIntosh ◽  
Geoffrey Y. Berguig ◽  
Omair A. Karim ◽  
Christa L. Cortesio ◽  
Rolando De Angelis ◽  
...  
Keyword(s):  
Light Scattering ◽  
Size Exclusion Chromatography ◽  
Molar Mass ◽  
Size Exclusion ◽  
Minimal Sample ◽  
Accuracy And Precision ◽  
Vector Genome ◽  
Exclusion Chromatography ◽  
Novel Applications

AbstractAdeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.

scholarly journals Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications

Nanoscale ◽  
2018 ◽  
Vol 10 (16) ◽  
pp. 7420-7430 ◽  
Author(s):  
Sean F. Gilmore ◽  
Timothy S. Carpenter ◽  
Helgi I. Ingólfsson ◽  
Sandra K. G. Peters ◽  
Paul T. Henderson ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Lipid Composition ◽  
High Density ◽  
Size Exclusion ◽  
High Density Lipoproteins ◽  
Serum Stability ◽  
Exclusion Chromatography

Nanolipoprotein assembly, and dissociation through contact with serum, as assessed through size-exclusion chromatography.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

Abstract 255: Plasminogen Promotes Cholesterol Efflux by the ABCA1

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Nathalie Pamir ◽  
David A Dichek ◽  
Godfrey S Getz ◽  
Santica Marcovina ◽  
Jay W Heinecke
Keyword(s):  
Size Exclusion Chromatography ◽  
Cholesterol Efflux ◽  
Particle Concentration ◽  
Specific Activity ◽  
Cholesterol Homeostasis ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Cvd Risk ◽  
Exclusion Chromatography

Background: The cholesterol efflux capacity (CEC) of serum HDL, measured using cultured macrophages predicts incident and prevalent CVD risk in humans. The ABCA1 pathway is a key regulator of macrophage cholesterol homeostasis in vivo. Methods: We used genetic and biochemical approaches in mice to identify important mediators of CEC. Results: On high-resolution size-exclusion chromatography of mouse plasma, macrophage CEC and HDL co-eluted as a single major peak, suggesting that HDL mediates cholesterol efflux. In contrast, size-exclusion chromatography revealed two major peaks of material that promoted ABCA1-specific CEC, one of which was distinct from HDL. HDL particle concentration was reduced by 75% in Apoa1 -/- mice; this resulted in a 50% decrease in macrophage CEC but, surprisingly, had no impact on ABCA1-specific CEC. Orthogonal chromatography-mass spectrometric analysis of the non-HDL-associated efflux inducing material isolated from wild-type and APOA1 deficient plasma showed that plasminogen strongly correlated with ABCA1-specific CEC. Moreover, isolated plasminogen promoted cholesterol efflux by the ABCA1 pathway, and the specific activity of ABCA1-specific CEC of non-HDL-associated material was reduced by 50% in plasminogen deficient plasma. Imaging of cells treated with fluorescently-labeled antibodies demonstrated that ABCA1 and plasminogen co-localized on the plasma membrane. Conclusions: HDL particle concentration is an important contributor to macrophage CEC. However, other pathways contribute to ABCA1-specific CEC; our studies identify plasminogen as one potential mediator. Plasminogen associates with CVD risk in human genetic studies, raising the possibility that it plays a role in atherosclerosis by modulating ABCA1-mediated sterol efflux from macrophages.

Measuring dissolved organic matter in estuarine and marine waters: size-exclusion chromatography with various detection methods

2018 ◽  
Vol 15 (7) ◽  
pp. 436 ◽  
Author(s):  
Gabriel Dulaquais ◽  
Johann Breitenstein ◽  
Matthieu Waeles ◽  
Rémi Marsac ◽  
Ricardo Riso
Keyword(s):  
Molecular Weight ◽  
Organic Matter ◽  
Organic Carbon ◽  
Dissolved Organic Matter ◽  
Humic Substances ◽  
Size Exclusion Chromatography ◽  
Size Exclusion ◽  
Marine Waters ◽  
Wide Range ◽  
Exclusion Chromatography

Environmental contextDissolved organic matter (DOM), a key parameter in aquatic biogeochemistry, is difficult to characterise owing to its variable composition and structure. We report a chromatographic method with carbon, nitrogen and absorbance detection able to record the size distribution of DOM and changes in its composition. The method could be used to identify additional sources to river or coastal waters as well as monitoring the DOM size/reactivity continuum in open oceans. AbstractWe studied the performance and limitations of size-exclusion chromatography with organic carbon, ultraviolet and organic nitrogen detectors (SEC-OCD-UVD-OND) for characterising dissolved organic matter (DOM) in estuarine and marine waters. We identified a strong salt effect on dissolved organic carbon (DOC) determination; however, calibration gave good results at salinity levels close to those of the sample analysed (ΔS ± 2 psu (practical salinity units)), with limited matrix effects, enabling an accurate measurement of DOC, as demonstrated by an intercalibration exercise. The repeatability, reproducibility and limit of detection (3 ppb for both carbon and nitrogen) for the three detectors demonstrated the robustness of the method for a wide range of natural waters, including carbon-rich freshwaters and deep seawaters with low carbon content (6000 ppb-C to 300 ppb-C). Deeper analysis of the SEC demonstrated that proteins and polysaccharides are partly fractionated within the column, and that terrestrial humic substances, isolated on a XAD-8 resin, can also be eluted in both fractions associated with biopolymers and low-molecular-weight neutrals. Application of the method to the study of DOM along a macrotidal estuary that was influenced by agricultural activities revealed significant changes in its composition despite a conservative DOC distribution. Distinct origins and qualities of high-molecular-weight (>500 kDa) organic compounds were identified for riverine and marine end-members. A new diagram to track changes in DOM lability is proposed to complete the humic-substances diagram.

scholarly journals Size and conformation of Ficoll as determined by size-exclusion chromatography followed by multiangle light scattering

2010 ◽  
Vol 298 (1) ◽  
pp. F205-F208 ◽  
Author(s):  
William H. Fissell ◽  
Christina L. Hofmann ◽  
Ross Smith ◽  
Michelle H. Chen
Keyword(s):  
Light Scattering ◽  
Size Exclusion Chromatography ◽  
Conformational Changes ◽  
Physiological Saline ◽  
Transport Processes ◽  
Size Exclusion ◽  
Random Coil ◽  
Filtration Barrier ◽  
Exclusion Chromatography

The characteristics of the glomerular filtration barrier (GFB) are challenging to measure, as macromolecular solutes in blood may be metabolized or transported by various cells in the kidney. Urinary solute concentrations generally reflect the cumulative influence of multiple transport processes rather than the intrinsic behavior of the GFB alone. Synthetic tracer molecules which are not secreted, absorbed, or modified by the kidney are useful tools. Ficoll, a globular polymer of epichlorohydrin and sucrose, is round, physiologically inert, and easily labeled, making it a nearly ideal glomerular probe. Fissell et al. reported filtration data suggesting that Ficoll was not as spherical as had been previously suggested (Fissell WH, Manley S, Dubnisheva A, Glass J, Magistrelli J, Eldridge AN, Fleischman AJ, Zydney AL, Roy S. Am J Physiol Renal Physiol 293: F1209–F1213, 2007). More recently, two investigators published comparisons of neutral and anionic Ficoll clearance that suggest Ficoll may undergo conformational changes when chemically derivatized (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Physiol 291: F1083–F1089, 2006; Guimaraes MAM, Nikolovski J, Pratt LM, Greive K, Comper WD. Am J Physiol Renal Physiol 285: F1118–F1124, 2003). To investigate Ficoll's characteristics further, we examined two commercial preparations, Ficoll 70 and Ficoll 400, by size-exclusion chromatography using a differential refractive index detector combined with light-scattering and viscosity detectors. A slope of 0.45 was obtained from the plot of the logarithm of molecular mass against the logarithm of root-mean square radius. The Mark-Houwink exponent values of 0.34 and 0.36 were calculated for Ficoll 70 and Ficoll 400, respectively. These results suggest Ficoll's conformation in physiological saline solution is likely intermediate between a solid sphere and a well-solvated linear random coil. The measurements help explain our previous observations and guide interpretation of in vivo experiments.

scholarly journals Size-Based Characterization of Freshwater Dissolved Organic Matter finds Similarities within a Water Body Type across Different Canadian Ecozones

2020 ◽  
Author(s):  
Pieter J. K. Aukes ◽  
Sherry L. Schiff ◽  
Jason J. Venkiteswaran ◽  
Richard J. Elgood ◽  
John Spoelstra
Keyword(s):  
Organic Matter ◽  
Dissolved Organic Matter ◽  
Size Exclusion Chromatography ◽  
Water Body ◽  
Size Exclusion ◽  
List Type ◽  
Body Type ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Dom Composition

ABSTRACTDissolved Organic Matter (DOM) represents a mixture of organic molecules that vary due to different source materials and degree of processing. Characterizing how DOM composition evolves along the aquatic continuum can be difficult. Using a size-exclusion chromatography technique (LC-OCD), we assessed the variability in DOM composition from both surface and groundwaters across a number of Canadian ecozones (mean annual temperature spanning −10 to +6 C). A wide range in DOM concentration was found from 0.2 to 120 mg C/L. Proportions of different size-based groupings across ecozones were variable, yet similarities between specific water-body types, regardless of location, suggest commonality in the processes dictating DOM composition. A PCA identified 70% of the variation in LC-OCD derived DOM compositions could be explained by the water-body type. We find that DOM composition within a specific water-body type is similar regardless of the differences in climate or surrounding vegetation where the sample originated from.HighlightsSize-exclusion chromatography (using LC-OCD) is a fast and effective tool to quantify differences in DOM composition across different environmentsProportions of biopolymers and low molecular weight fractions can distinguish between surface and groundwater DOMSimilar water-body types have comparable DOM size compositions across ecozones that range in annual air temperatures from –10 to 6ºC

scholarly journals Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Diána Kitka ◽  
Judith Mihály ◽  
Jean-Luc Fraikin ◽  
Tamás Beke-Somfai ◽  
Zoltán Varga
Keyword(s):  
Size Distribution ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Fluorescence Detection ◽  
New Technology ◽  
Size Exclusion ◽  
Purification Technique ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Ev Model

AbstractNew methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

scholarly journals Metal-Polymer Complexes of Gallium/Gallium-68 with Copolymers of N-Vinylpyrrolidonewith N-Vinylformamideand N-Vinyliminodiacetic Acid: A Hint for Radiolabeling of Water-Soluble Synthetic Flexible Chain Macromolecules

Polymers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 2889
Author(s):  
Nikolay I. Gorshkov ◽  
Аndrey Yu. Murko ◽  
Iirina I. Gavrilova ◽  
Мarina А. Bezrukova ◽  
Аlbert I. Kipper ◽  
...  
Keyword(s):  
Water Soluble ◽  
Size Exclusion ◽  
Optical Methods ◽  
Polymer Complex ◽  
Polymer Complexes ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Gallium 68 ◽  
Metal Polymer

Copolymer of N-vinylpyrrolidone (VP) with vinylformamide (VFA) and N-vinyliminodiacetic acid (VIDA) was synthesized; its metal-polymer complexes (MPCs) with gallium were obtained. The complexes were characterized by size exclusion chromatography, hydrodynamic and optical methods, scanning electron microscopy, and spectral methods (UV, IR, 1Н NMR spectroscopy). It was demonstrated that in going from polymer to complex, hydrodynamic parameters of macromolecules change only slightly, although the polymer contains intramolecular Ga(VIDA)2 fragments in its structure. A new method for preparation of MPCs with gallium and gallium-68 radionuclide was suggested. The obtained metal-polymer complex is stable over a wide range of pH values as well as in the histidine challenge reaction. In vivo distribution experiments in intact animals showed high primary accumulation of thegallium-68 MPC in blood with subsequent excretion via urinary tract.

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