scholarly journals Methylated Trivalent Arsenic-Glutathione Complexes are More Stable than their Arsenite Analog

2008 ◽  
Vol 2008 ◽  
pp. 1-8 ◽  
Author(s):  
Andrew J. Percy ◽  
Jürgen Gailer
Keyword(s):  
Arsenic Species ◽  
Size Exclusion ◽  
Trivalent Arsenic ◽  
Physiological Conditions ◽  
Column Formation ◽  
Monomethylarsonous Acid ◽  
Exclusion Chromatography ◽  
Dimethylarsinous Acid ◽  
Phosphate Buffered Saline ◽  
Comparative Stability

The trivalent arsenic glutathione complexes arsenic triglutathione, methylarsonous diglutathione, and dimethylarsinous glutathione are key intermediates in the mammalian metabolism of arsenite and possibly represent the arsenic species that are transported from the liver to the kidney for urinary excretion. Despite this, the comparative stability of the arsenic-sulfur bonds in these complexes has not been investigated under physiological conditions resembling hepatocyte cytosol. Using size-exclusion chromatography and a glutathione-containing phosphate buffered saline mobile phase (5 or 10 mM glutathione, pH 7.4) in conjunction with an arsenic-specific detector, we chromatographed arsenite, monomethylarsonous acid, and dimethylarsinous acid. The on-column formation of the corresponding arsenic-glutathione complexes between 4 and37°C revealed that methylated arsenic-glutathione complexes are more stable than arsenic triglutathione. The relevance of these results with regard to the metabolic fate of arsenite in mammals is discussed.

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.

Unstable trivalent arsenic metabolites, monomethylarsonous acid and dimethylarsinous acid

2001 ◽  
Vol 16 (12) ◽  
pp. 1409-1413 ◽  
Author(s):  
Zhilong Gong ◽  
Xiufen Lu ◽  
William R. Cullen ◽  
X. Chris Le
Keyword(s):  
Trivalent Arsenic ◽  
Monomethylarsonous Acid ◽  
Dimethylarsinous Acid ◽  
Arsenic Metabolites

New insights into change of lens proteins’ stability with ageing under physiological conditions

2021 ◽  
pp. bjophthalmol-2021-319834
Author(s):  
Chenqi Luo ◽  
Jingjie Xu ◽  
Chenxi Fu ◽  
Ke Yao ◽  
Xiangjun Chen
Keyword(s):  
Protein Stability ◽  
Size Exclusion Chromatography ◽  
Therapeutic Option ◽  
Size Exclusion ◽  
Physiological Status ◽  
Lens Protein ◽  
Lens Proteins ◽  
Physiological Conditions ◽  
Age Related ◽  
Exclusion Chromatography

BackgroundAge-related cataract, which presents as a cloudy lens, is the primary cause of vision impairment worldwide and can cause more than 80% senile blindness. Previous studies mainly explored the profile of lens proteins at a low concentration because of technical limitations, which could not reflect physiological status. This study focuses on protein stability changes with ageing under physiological conditions using a novel equipment, Unchained Labs (Uncle), to evaluate protein thermal stability.MethodsSamples were assessed through Unchained Labs, size-exclusion chromatography, western blot and biophysics approaches including the Thioflavin T, ultraviolet and internal fluorescence.ResultsWith age, the melting temperature value shifted from 67.8°C in the young group to 64.2°C in the aged group. Meanwhile, crystallin may form more isomeric oligomers and easy to be degraded in aged lenses. The spectroscopic and size-exclusion chromatography results show a higher solubility after administrated with lanosterol under the environmental stress.ConclusionWe are the first to explore rabbit lens protein stability changes with ageing using biophysical methods under physiological conditions, and this study can conclude that the structural stability and solubility of lens proteins decrease with ageing. Additionally, lanosterol could aid in resolving protein aggregation, making it a potential therapeutic option for cataracts. So, this study provides cataract models for anti-cataract drug developments

scholarly journals Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study

2021 ◽  
Vol 22 (2) ◽  
pp. 979
Author(s):  
Carmen Almiñana ◽  
Alba Rudolf Vegas ◽  
Muhittin Tekin ◽  
Mubbashar Hassan ◽  
Rustem Uzbekov ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Optimal Method ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Uterine Fluid ◽  
Exclusion Chromatography ◽  
Protein Classes ◽  
Uterine Lavage ◽  
Phosphate Buffered Saline

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

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.

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

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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