Fractional Clearance of Albumin is Influenced by its Degradation during Renal Passage

1997 ◽  
Vol 93 (6) ◽  
pp. 557-564 ◽  
Author(s):  
Tanya M. Osicka ◽  
Sianna Panagiotopoulos ◽  
George Jerums ◽  
Wayne D. Comper
Keyword(s):  
Size Exclusion ◽  
Gel Chromatography ◽  
Small Peptides ◽  
Isolated Perfused Kidney ◽  
A Value ◽  
Radioactivity Analysis ◽  
Fractional Clearance ◽  
Exclusion Chromatography ◽  
Perfused Kidney

1. The fractional clearance of intact albumin as determined by fractionation of urine by gel chromatography gave a value of 3.9 ± 1.6 × 10−4 for the isolated perfused kidney and 2.1 ± 0.6 × 10−4in vivo using ALZET osmotic pumps. 2. Albumin fractional clearance as measured by detection of the tritium label on the albumin molecule by radioactivity analysis gave a value of 7.5 ± 3.9 × 10−3 for the isolated perfused kidney and 2.3 ± 0.9 × 10−3in vivo. 3. The major differences between assays that detect intact albumin compared with non-specific assays in the estimates of the fractional clearance of albumin can be explained by the degradation of approx. 90% of albumin to small peptides during its renal passage. This has been demonstrated by size exclusion chromatography of urine samples from experiments where (i) exogenous tritium-labelled albumin was used in isolated perfused kidneys, (ii) exogenous tritium-labelled albumin was administered intravenously and (iii) analysis was made with metabolically labelled endogenous albumin in vivo.

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

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.

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 Fibrillar pharmacology of functionalized nanocellulose

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.

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.

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

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
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.

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.

Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins

1994 ◽  
Vol 140 (3) ◽  
pp. 475-482 ◽  
Author(s):  
A P D Lord ◽  
S E P Bastian ◽  
L C Read ◽  
P E Walton ◽  
F J Ballard
Keyword(s):  
Binding Proteins ◽  
Rat Plasma ◽  
Size Exclusion ◽  
Free Form ◽  
Igf Binding Proteins ◽  
Igf I ◽  
Exclusion Chromatography ◽  
Igf Binding

Abstract Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1–3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep>human>pig=chicken>rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig=chicken>sheep>human>rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1–3)IGF-I binding, which in turn was greater than LR3 IGF-I binding. These experiments suggest first that IGF-binding properties measured after the removal of endogenous IGFs do not always reflect the situation with untreated plasma or in vivo, and secondly, the increased potencies of des(1–3)IGF-I and LR3 IGF-I in rat growth studies that have been ascribed to higher concentrations of these peptides in the free form cannot necessarily be extended to other species. Journal of Endocrinology (1994) 140, 475–482

scholarly journals Melanogenesis: A Search for Pheomelanin and Also, What Is Lurking Behind Those Dark Colors?

2019 ◽  
Author(s):  
Koen Vercruysse ◽  
Venise Govan
Keyword(s):  
Size Exclusion ◽  
Black Pigment ◽  
Main Reaction ◽  
Main Reaction Product ◽  
Light Yellow ◽  
Exclusion Chromatography ◽  
Co Precipitation ◽  
Mediated Oxidation

<p>We investigated the synthesis of melanin-like materials from DOPA, dopamine, norepinephrine and epinephrine in the presence of L-cysteine. We observed that L-cysteine delayed the formation of pigment from these catecholamines and that the presence of L-cysteine yielded darker-colored reaction mixtures. No reddish pigment was observed that would indicate the synthesis of pheomelanin-like material. The reactions were performed in the presence of Na<sub>2</sub>CO<sub>3</sub> and through the addition of CaCl<sub>2</sub> at the end of the reaction; the black, eumelanin-like material was co-precipitated with CaCO<sub>3</sub>. The remaining supernatant solutions were observed to be light-yellow to rusty-orange in color depending on the catecholamine used in the reaction. Size exclusion chromatography (SEC) analyses indicated that the removal of the black pigment left behind an oligomeric material that exhibited a strong absorbance band around 280nm. Our experimental and analytical observations prompt us to raise a number of points of discussion or hypotheses. 1) The presence of L-cysteine during the air-mediated oxidation of catecholamines leads to darker-colored pigments; not reddish or lighter-colored pigments that would visually resemble pheomelanin-like pigments, 2) SEC analyses suggested that the black pigment generated during the air-mediated oxidation of catecholamines is not necessarily the main reaction product, 3) The pre-formed, dark-colored pigments obtained through the air-mediated oxidative melanogenesis process can readily be deposited on insoluble mineral surfaces using an <i>in situ</i> co-precipitation procedure, 4) The air-mediated oxidation of catecholamines leads to a binary product that contains an insoluble, melanin-like substance and a soluble, oligo- or polymeric substance containing unoxidized precursor units, 5) The melanogenesis process leads to a binary product involving a non-covalently bonded combination of dark-colored pigment and a lighter-colored or colorless substance; the latter being understudied or ignored in the <i>in vitro</i> or <i>in vivo</i> studies of the melanogenesis process, 6) The kinetics of the melanogenesis process may determine the balance between insoluble and soluble components of the binary product generated; the slower the reaction the more dark-colored, insoluble pigment generated, 7) One should consider the possibility of intermolecularly, N-to-C, bonded units of catecholamines when evaluating the structure of melanins, polydopamines, etc. and 8) There is a need for a systematic study of the effect of amino acids (beyond just L-cysteine) and amines in general on the melanogenesis process.</p>

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 415: Role of Proteolysis in the Remodeling of High-Density Lipoprotein

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Shobini Jayaraman
Keyword(s):  
Western Blotting ◽  
Density Lipoprotein ◽  
Cell Types ◽  
Size Exclusion ◽  
Major Protein ◽  
Physiological Conditions ◽  
Hdl Functionality ◽  
Exclusion Chromatography ◽  
Arterial Intima

Introduction: Both quantity and quality of the circulating HDL determine their optimal anti-atherogenic potential. During atherogenesis, various cell types in the arterial intima release enzymes into the intimal fluid, which modify HDL proteins and lipids that adversely affect HDL functionality. Hypothesis: The emerging paradigm for the in vivo proteolytic inactivation of HDL is centered on pre-beta-HDL. Over 90% of the major HDL proteins, apoA-I and apoA-II, circulate on mature HDL. Although binding to HDL protects these proteins from proteolysis, such proteolysis cannot be completely excluded, and its effects on HDL functionality remain unknown. Methods: Human plasma HDL were subjected to mild proteolysis with plasmin, a protease active in atherosclerotic lesions. The proteolytic products were analyzed by SDS-PAGE and Western blotting. HDL remodeling was monitored under near-physiological conditions by size-exclusion chromatography and gel electrophoresis. Results: HDL treatment with plasmin caused no significant structural remodeling of lipoprotein particles. Interestingly, plasmin cleaved apoA-I and apoA-II on HDL. The major protein fragments were observed in the 10-12 kDa range. Western blotting indicated that these fragments were derived from both apoA-I and apoA-II. Next, intact and plasmin-treated HDL were incubated at 37 o C, pH 7.5 for 6-12 h. Intact HDL showed dissociation of a fraction of lipid-free apoA-I without significant changes in the particle size. In contrast, plasmin-treated HDL underwent fusion with release of full-length and fragmented apoA-I and apoA-II, indicating lipoprotein destabilization. Conclusion: Our results reveal that plasmin, can cleave HDL-bound forms of apoA-I and apoA-II and thereby destabilize HDL under near-physiological conditions, resulting in HDL disintegration and dissociation of lipid-free proteins. For the first time, we demonstrate that proteolysis can render not only lipid-free but also HDL-bound proteins dysfunctional. Destabilization of HDL via proteolytic modifications may contribute to the recently observed excessive accumulation of lipid-free apoA-I in the arterial intima, which probably contributes to the progression of atherosclerosis.

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