scholarly journals Effects of 3FTx Protein Fraction from Naja ashei Venom on the Model and Native Membranes: Recognition and Implications for the Mechanisms of Toxicity

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2164
Author(s):  
Barbara Dyba ◽  
Elżbieta Rudolphi-Szydło ◽  
Anna Barbasz ◽  
Agnieszka Czyżowska ◽  
Konrad Kamil Hus ◽  
...  
Keyword(s):  
Protein Fraction ◽  
Therapeutic Potential ◽  
Langmuir Monolayers ◽  
Structural Response ◽  
The Body ◽  
Size Exclusion ◽  
Mechanisms Of Toxicity ◽  
Unsaturated Lipids ◽  
Exclusion Chromatography ◽  
Polar Parts

Three-finger toxins are naturally occurring proteins in Elapidae snake venoms. Nowadays, they are gaining popularity because of their therapeutic potential. On the other hand, these proteins may cause undesirable reactions inside the body′s cells. A full assessment of the safety of Naja ashei venom components for human cell application is still unknown. The aim of the study was to determine the effect of the exogenous application of three-finger toxins on the cells of monocytes (U-937) and promyelocytes (HL-60), with particular emphasis on the modification of their membranes under the influence of various doses of 3FTx protein fraction (0–120 ng/mL). The fraction exhibiting the highest proportion of 3FTx proteins after size exclusion chromatography (SEC) separation was used in the experiments. The structural response of cell membranes was described on the basis of single-component and multi-component Langmuir monolayers that mimicked the native membranes. The results show that the mechanism of protein–lipid interactions depends on both the presence of lipid polar parts (especially zwitterionic type of lipids) and the degree of membrane saturation (the greatest-for unsaturated lipids). The biochemical indicators reflecting the tested cells (MDA, LDH, cell survival, induction of inflammation, LD50) proved the results that were obtained for the model.

scholarly journals Identification of food-derived peptides in human blood after ingestion of corn and wheat gluten hydrolysates

2018 ◽  
Vol 2 ◽  
Author(s):  
Akika Ejima ◽  
Megumi Nakamura ◽  
Yasushi A. Suzuki ◽  
Kenji Sato
Keyword(s):  
Matrix Protein ◽  
Leucine Aminopeptidase ◽  
Wheat Gluten ◽  
Protein Hydrolysates ◽  
The Body ◽  
Size Exclusion ◽  
Extracellular Matrix Protein ◽  
Reverse Phase Hplc ◽  
Before And After ◽  
Exclusion Chromatography

Bioactive peptides in the body after ingestion of plant protein hydrolysates have been speculated but not identified. We aimed to establish an approach to identify small amounts of food-derived peptides in humans after ingestion of non-extracellular matrix protein hydrolysates. Corn and wheat gluten hydrolysates were digested using pancreatin and leucine aminopeptidase; the resultant peptides were identified via size-exclusion chromatography and reverse-phase HPLC-tandem mass spectrometry (MS/MS). Structures of indigestible peptides were confirmed via LC-MS/MS in multi-reaction monitoring mode. All indigestible peptides in the exopeptidase digest were diprolyl and di- and tripyroglutamyl peptides. Blood collected from healthy volunteers (n = 4) before and after ingestion of 9 g of the hydrolysates was assessed for the indigestible peptides via LC-MS/MS. Six peptides (Pro-Ala, Pro-Gly, Pro-Gln, pyroGlu-Pro, pyroGlu-Leu-Pro, and pyroGlu-Gln-Pro) significantly increased in human plasma up to 10–100 nM compared to the baseline. This may hence be a powerful tool for identifying foodderived peptides in blood.

scholarly journals High-grade extracellular vesicles preparation by combined size-exclusion and affinity chromatography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cristina Bellotti ◽  
Kristina Lang ◽  
Nataliya Kuplennik ◽  
Alejandro Sosnik ◽  
Robert Steinfeld
Keyword(s):  
Affinity Chromatography ◽  
Extracellular Vesicles ◽  
Biological Function ◽  
Therapeutic Potential ◽  
Folate Receptor ◽  
Size Exclusion ◽  
Selective Enrichment ◽  
Large Numbers ◽  
Marker Proteins ◽  
Exclusion Chromatography

AbstractExtracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.

scholarly journals Comprehensive Profiling of Plasma Exosomes Using Data-Independent Acquisitions – New Tools for Aging Cohort Studies

2021 ◽  
Author(s):  
Sandip K. Patel ◽  
Roland Bruderer ◽  
Nathan Basisty ◽  
Joanna Bons ◽  
Pierre-Yves Desprez ◽  
...  
Keyword(s):  
Reversed Phase ◽  
The Body ◽  
Size Exclusion ◽  
Biomarkers Of Aging ◽  
Age Related ◽  
Complex Biological Process ◽  
High Throughput Method ◽  
Aging Study ◽  
Exclusion Chromatography ◽  
Using Data

AbstractAging is a complex biological process associated with progressive loss of physiological function and susceptibility to several diseases, such as cancer and neurodegeneration. Exosomes are involved in many cellular signaling pathways, and their cargo may serve as promising disease or aging biomarkers. These membrane-bound extracellular vesicles facilitate the transport of intracellular contents to proximal and distal cells in the body. Here, we investigated two omics approaches for exosome analysis. To overcome the challenges of plasma exosome contamination with abundant soluble plasma proteins, we developed a high-throughput method to isolate highly purified exosomes from human plasma by sequential size-exclusion chromatography and ultrafiltration. First, we used data-dependent acquisitions from offline high-pH reversed-phase fractions of exosome lysate to generate a deep spectral library comprising ∼2,300 exosome proteins. Second, in a pilot aging study, we used comprehensive data-independent acquisitions to compare plasma exosomes from young (20–26 yrs) and old (60–66 yrs) individuals. We quantified 1,318 exosome proteins, and levels of 144 proteins were significantly different in young and old plasma groups (Q<0.05 and >1.5-fold change). We also analyzed exosome miRNA cargo and detected 331 miRNAs. Levels of several were significantly different in young and old individuals. In addition, 88 and 17 miRNAs were unique to old and young individuals, respectively. Plasma exosome biomarkers have great potential for translational studies investigating biomarkers of aging and age-related diseases and to monitor therapeutic aging interventions.

scholarly journals Selenoproteome Expression Studied by Non-Radioactive Isotopic Selenium-Labeling in Human Cell Lines

2021 ◽  
Vol 22 (14) ◽  
pp. 7308
Author(s):  
Jordan Sonet ◽  
Anne-Laure Bulteau ◽  
Zahia Touat-Hamici ◽  
Maurine Mosca ◽  
Katarzyna Bierla ◽  
...  
Keyword(s):  
Inductively Coupled Plasma ◽  
The Body ◽  
Size Exclusion ◽  
Selenium Atom ◽  
Western Blots ◽  
Selective Labeling ◽  
Inductively Coupled ◽  
Exclusion Chromatography ◽  
Plasma Mass Spectrometry ◽  
Selenium Isotopes

Selenoproteins, in which the selenium atom is present in the rare amino acid selenocysteine, are vital components of cell homeostasis, antioxidant defense, and cell signaling in mammals. The expression of the selenoproteome, composed of 25 selenoprotein genes, is strongly controlled by the selenium status of the body, which is a corollary of selenium availability in the food diet. Here, we present an alternative strategy for the use of the radioactive 75Se isotope in order to characterize the selenoproteome regulation based on (i) the selective labeling of the cellular selenocompounds with non-radioactive selenium isotopes (76Se, 77Se) and (ii) the detection of the isotopic enrichment of the selenoproteins using size-exclusion chromatography followed by inductively coupled plasma mass spectrometry detection. The reliability of our strategy is further confirmed by western blots with distinct selenoprotein-specific antibodies. Using our strategy, we characterized the hierarchy of the selenoproteome regulation in dose–response and kinetic experiments.

scholarly journals High-grade Extracellular Vesicles Preparation by Combined Size- exclusion and Affinity Chromatography

2021 ◽  
Author(s):  
Cristina Bellotti ◽  
Kristina Lang ◽  
Nataliya Kuplennik ◽  
Alejandro Sosnik ◽  
Robert Steinfeld
Keyword(s):  
Affinity Chromatography ◽  
Extracellular Vesicles ◽  
Biological Function ◽  
Therapeutic Potential ◽  
Folate Receptor ◽  
Size Exclusion ◽  
Selective Enrichment ◽  
Large Numbers ◽  
Marker Proteins ◽  
Exclusion Chromatography

Abstract Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes, microvesicles, microparticles, and ectosomes which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.

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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