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

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

International Journal of Molecular Sciences ◽

10.3390/ijms22094512 ◽

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.

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Fibrillar pharmacology of functionalized nanocellulose

Scientific Reports ◽

10.1038/s41598-020-79592-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sam Wong ◽

Simone Alidori ◽

Barbara P. Mello ◽

Bryan Aristega Almeida ◽

David Ulmert ◽

...

Keyword(s):

Aspect Ratio ◽

Fluorescent Dyes ◽

Pharmacokinetic Profile ◽

Water Soluble ◽

Size Exclusion ◽

Hydroxyl Groups ◽

Target Tissue ◽

Exclusion Chromatography ◽

Specific Accumulation

AbstractCellulose nanocrystals (CNC) are linear organic nanomaterials derived from an abundant naturally occurring biopolymer resource. Strategic modification of the primary and secondary hydroxyl groups on the CNC introduces amine and iodine group substitution, respectively. The amine groups (0.285 mmol of amine per gram of functionalized CNC (fCNC)) are further reacted with radiometal loaded-chelates or fluorescent dyes as tracers to evaluate the pharmacokinetic profile of the fCNC in vivo. In this way, these nanoscale macromolecules can be covalently functionalized and yield water-soluble and biocompatible fibrillar nanoplatforms for gene, drug and radionuclide delivery in vivo. Transmission electron microscopy of fCNC reveals a length of 162.4 ± 16.3 nm, diameter of 11.2 ± 1.52 nm and aspect ratio of 16.4 ± 1.94 per particle (mean ± SEM) and is confirmed using atomic force microscopy. Size exclusion chromatography of macromolecular fCNC describes a fibrillar molecular behavior as evidenced by retention times typical of late eluting small molecules and functionalized carbon nanotubes. In vivo, greater than 50% of intravenously injected radiolabeled fCNC is excreted in the urine within 1 h post administration and is consistent with the pharmacological profile observed for other rigid, high aspect ratio macromolecules. Tissue distribution of fCNC shows accumulation in kidneys, liver, and spleen (14.6 ± 6.0; 6.1 ± 2.6; and 7.7 ± 1.4% of the injected activity per gram of tissue, respectively) at 72 h post-administration. Confocal fluorescence microscopy reveals cell-specific accumulation in these target tissue sinks. In summary, our findings suggest that functionalized nanocellulose can be used as a potential drug delivery platform for the kidneys.

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Water soluble metal-polymeric complexes of gallium and triple co-polymer of N-vinylpyrrolidone with N-vinylformamide and N-vinylminodiacetic acid

Доклады Академии наук ◽

10.31857/s0869-56524853306-310 ◽

2019 ◽

Vol 485 (3) ◽

pp. 306-310

Author(s):

N. I. Gorshkov ◽

A. Yu. Murko ◽

I. I. Gavrilova ◽

I. I. Malakhova ◽

V. D. Krasikov ◽

...

Keyword(s):

Molecular Weight ◽

1H Nmr ◽

Metal Ion ◽

Water Soluble ◽

Polymer Complex ◽

Nmr Data ◽

Exclusion Chromatography ◽

Polymeric Complexes ◽

Metal Polymer

A terpolymer with a molecular weight of 45 kDa containing 7 mol.% of vinylamine units, 80 mol.% of vinylpyrrolidone, and 3 mol.% of vinyliminodiacetic acid units has been synthesized. Its complexation with Ga3+ ion has been studied by HPLC. The resulting metal–polymer complex has been characterized by exclusion chromatography and spectral (IR, 1H NMR) data. The complex has a monomolecular structure where the metal ion acts as an anchor fragment between vinyliminodiacetic acid units and is stable in the reaction of interligand exchange with histidine.

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CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

Clinical & Investigative Medicine ◽

10.25011/cim.v32i6s.11137 ◽

2009 ◽

Vol 32 (6S) ◽

pp. 3

Author(s):

A Baass ◽

H Wassef ◽

M Tremblay ◽

L Bernier ◽

R Dufour ◽

...

Keyword(s):

Modifier Gene ◽

Size Exclusion ◽

Plasma Lipoproteins ◽

Lipoprotein Profile ◽

Exclusion Chromatography ◽

Low Hdl ◽

Lcat Deficiency ◽

Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

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Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris

10.26686/wgtn.12597809.v1 ◽

2020 ◽

Author(s):

M Wee ◽

M Mastrangelo ◽

Susan Carnachan ◽

Ian Sims ◽

K Goh

Keyword(s):

New Zealand ◽

Uronic Acid ◽

Average Molecular Weight ◽

Shear Thickening ◽

Water Soluble ◽

Size Exclusion ◽

Resonance Spectroscopy ◽

Tree Fern ◽

13C Nuclear Magnetic Resonance ◽

Exclusion Chromatography

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ~1.9×106Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure. © 2014 Elsevier B.V.

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Human Plasma and Recombinant Hemopexins: Heme Binding Revisited

International Journal of Molecular Sciences ◽

10.3390/ijms22031199 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1199

Author(s):

Elena Karnaukhova ◽

Catherine Owczarek ◽

Peter Schmidt ◽

Dominik J. Schaer ◽

Paul W. Buehler

Keyword(s):

Protein S ◽

Soret Band ◽

Cell System ◽

Size Exclusion ◽

Heme Binding ◽

Systematic Analysis ◽

Molar Ratios ◽

Recombinant Production ◽

Wide Range ◽

Exclusion Chromatography

Plasma hemopexin (HPX) is the key antioxidant protein of the endogenous clearance pathway that limits the deleterious effects of heme released from hemoglobin and myoglobin (the term “heme” is used in this article to denote both the ferrous and ferric forms). During intra-vascular hemolysis, heme partitioning to protein and lipid increases as the plasma concentration of HPX declines. Therefore, the development of HPX as a replacement therapy during high heme stress could be a relevant intervention for hemolytic disorders. A logical approach to enhance HPX yield involves recombinant production strategies from human cell lines. The present study focuses on a biophysical assessment of heme binding to recombinant human HPX (rhHPX) produced in the Expi293FTM (HEK293) cell system. In this report, we examine rhHPX in comparison with plasma HPX using a systematic analysis of protein structural and functional characteristics related to heme binding. Analysis of rhHPX by UV/Vis absorption spectroscopy, circular dichroism (CD), size-exclusion chromatography (SEC)-HPLC, and catalase-like activity demonstrated a similarity to HPX fractionated from plasma. In particular, the titration of HPX apo-protein(s) with heme was performed for the first time using a wide range of heme concentrations to model HPX–heme interactions to approximate physiological conditions (from extremely low to more than two-fold heme molar excess over the protein). The CD titration data showed an induced bisignate CD Soret band pattern typical for plasma and rhHPX versions at low heme-to-protein molar ratios and demonstrated that further titration is dependent on the amount of protein-bound heme to the extent that the arising opposite CD couplet results in a complete inversion of the observed CD pattern. The data generated in this study suggest more than one binding site in both plasma and rhHPX. Furthermore, our study provides a useful analytical platform for the detailed characterization of HPX–heme interactions and potentially novel HPX fusion constructs.

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

Scientific Reports ◽

10.1038/s41598-021-82599-1 ◽

2021 ◽

Vol 11 (1) ◽

Cited By ~ 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.

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Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

Biochemical Journal ◽

10.1042/bj3500139 ◽

2000 ◽

Vol 350 (1) ◽

pp. 139-147 ◽

Cited By ~ 11

Author(s):

Diego F. GÓMEZ CASATI ◽

Miguel A. AON ◽

Alberto A. IGLESIAS

Keyword(s):

Conformational Changes ◽

Tertiary Structure ◽

Fluorescence Emission ◽

Maximal Activity ◽

Size Exclusion ◽

Exclusion Chromatography ◽

Adp Glucose Pyrophosphorylase ◽

Kinetics Of ◽

Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

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Synthesis of Water-Soluble Copolymers of N-vinylpyrrolidone with N-vinyldithiocarbamate as Multidentate Polymeric Chelation Systems and Their Complexes with Indium and Gallium

Molecules ◽

10.3390/molecules25204681 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4681

Author(s):

Nikolay I. Gorshkov ◽

Andrey Yu. Murko ◽

Irina I. Gavrilova ◽

Marina A. Bezrukova ◽

Albert I. Kipper ◽

...

Keyword(s):

Metal Ions ◽

Alkaline Medium ◽

Carbon Disulfide ◽

Water Soluble ◽

Polymer Complexes ◽

Chromatographic Methods ◽

Gallium Metal ◽

Molecular Masses ◽

Derivatives Of ◽

Metal Polymer

Dithiocarbamate (DTC) derivatives of N-vinylpyrrolidone-N-vinylamine (VP–VA) copolymers were synthesized via reaction between the copolymers and carbon disulfide in alkaline medium; molecular masses of the products were 12 and 29 kDa; the VP:VDTC ratios were 94:6 and 83:17 mol.%. Complexation between the obtained DTC derivatives and metal ions (indium and gallium) was investigated. It was demonstrated that metal–DTC ligand complexes with 1:3 ratio between components were formed. Gallium metal–polymer complexes (MPC) were unstable in solution. Individual indium MPC were isolated and characterized by spectral and chromatographic methods. Unlike similar gallium MPC, they appeared to be stable in histidine challenge reaction.

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